Pro- and antidegenerative effects of JNK stresskinases in ... · 4.3.2 Activation of the mitochondrial pool of JNK following 6-OHDA 98 4.3.3 Translocation of c-Jun N-terminal kinase

Pro- and antidegenerative effects of JNK stresskinases

in neuronal cells

Dissertation

zur Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät

der Christian-Albrechts-Universität

zu Kiel

vorgelegt von

Lutz Römer

Kiel, 2008

Deutscher Titel: Pro- und antidegenerative Wirkungen der JNK Stresskinasen in neuronalen Zellen

Referent: Prof. Dr. Thomas Herdegen

Koreferent: Prof. Dr. Eric Beitz

Tag der mündlichen Prüfung: 04.06.2008

Zum Druck genehmigt: 04.06.2008

“I believe that unarmed truth and unconditional love will have the final word in reality. This is why right, temporarily defeated, is stronger than evil triumphant.” Martin Luther King, Jr.

TABLE OF CONTENTS

5

TABLE OF CONTENTS

1. INTRODUCTION 9

1.1 Parkinson’s disease (PD) 9

1.1.1 Current treatment of PD 9

1.1.2 PD in clinical research 10

1.1.3 Experimental models of PD 10

1.1.3.1 6-hydroxydopamine 11

1.1.3.2 MPTP 12

1.1.4 Genetic factors and implications for idiopathic PD 13

1.2 Oxidative stress 13

1.2.1 Antioxidant defense 16

1.2.2 Role of ROS and ROS products in cell death 18

1.3 Hallmarks of apoptosis 20

1.4 Mitochondria in neuronal cell death 20

1.5 MAPK cascades 23

1.5.1 JNK 24

2. AIMS OF THE THESIS 27

3. MATERIALS AND METHODS 28

3.1 Materials 28

3.2 Laboratory Equipment 32

3.3 Methods 33

3.3.1 PC12 cell culture 33

3.3.1.1 Splitting 34

3.3.1.2 Freezing and thawing 34

3.3.2 Applied stimuli 35

3.3.2.1 6-hydroxydopamine (6-OHDA) 35

3.3.2.2 1-Methyl-4-phenylpyridinium ion (MPP+) 35

3.3.2.3 Staurosporine (STS) 36

3.3.2.4 Valinomycin 37

3.3.3 Protection of PC12 cells 37

3.3.3.1 The JNK inhibitor SP600125 37

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3.3.3.2 Methysticin 37

3.3.3.3 Luteolin 39

3.3.3.4 Resveratrol 39

3.3.3.5 tert-butylhydroquinone 40

3.3.4 Trypan blue viability assay 40

3.3.5 Preparation of mitochondria 40

3.3.6 Protein identification by Western blot 42

3.3.6.1 Denaturing protein extraction 42

3.3.6.2 SDS-PAGE 42

3.3.6.3 Preparation of polyacrylamide gels 43

3.3.6.4 Preparation of protein samples 45

3.3.6.5 Electrophoresis 45

3.3.6.6 Immunoblotting 45

3.3.6.7 ECL-reaction 47

3.3.6.8 Stripping of Western blot membranes 47

3.3.6.9 Ponceau S staining of Western blot membranes 48

3.3.7 Basic principles of flow cytometry 48

3.3.7.1 Flow cytometrical data analysis 49

3.3.7.2 Staining of PC12 cells with ROS-sensitive fluorescent dyes 50

3.3.7.2.1 2’,7’-Dichlorofluorescein (DCF) 50

3.3.7.2.2 Dihydrorhodamine (DHR) 51

3.3.7.3 Assessment of the mitochondrial membrane potential with JC-1 51

3.3.7.4 Detection of apoptosis and necrosis in the flow cytometer 54

3.3.8 Experiments using the fluorescence microplate reader 56

3.3.9 Experiments using the spectrofluorometer 58

3.3.10 Fluorescence microscopy 59

3.3.10.1 Principle of confocal laser scanning microscopy 59

3.3.10.2 Experimental setup 60

3.3.11 Breeding of mice 61

3.3.12 Genetic characterization of JNK knock-out mice strains 61

3.3.12.1 Polymerase chain reaction (PCR) 62

3.3.12.2 Detection and analysis of the PCR reaction product 64

3.3.13 Isolation of mitochondria from mice brain 65

3.3.13.1 Determination of mitochondrial proteins 66

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3.3.13.2 Estimation of the quality of isolated mitochondria measuring the respiratory control ratio (RCR)

67

3.3.13.3 Purity of mitochondrial preparations 69

3.3.14 Primary cells 69

3.3.14.1 Coating of the plates 70

3.3.14.2 Obtaining and cultivating primary murine neurons 70

3.3.14.3 Treatment of primary cells 71

3.3.14.4 Immunocytochemistry 71

3.3.15 Statistical analysis 72

4. RESULTS 73

4.1 Mechanisms of 6-hydroxydopamine mediated cell death 74

4.1.1 6-hydroxydopamine induces cell death in PC12 cells 74

4.1.2 6-hydroxydopamine generates reactive oxygen species in PC12 cells 744.1.3 The mitochondrial membrane potential collapses after 6-hydroxy-

dopamine treatment 80

4.1.4 6-hydroxydopamine induces cytochrome c release 89

4.2 Responsiveness of isolated mitochondria to 6-hydroxydopamine 90

4.2.1 Analyses with isolated mitochondria 90

4.2.2 Quality of isolated mitochondria 90

4.2.3 Purity of mitochondrial fractions 91

4.2.4 ROS levels in isolated mitochondria following 6-hydroxydopamine 914.2.5 The mitochondrial membrane potential of isolated mitochondria

following 6-OHDA 92

4.3 The mitochondrial death pathway and c-Jun N-terminal kinase (JNK) signaling

97

4.3.1 Purification of PC12 cell mitochondrial fractions 97

4.3.2 Activation of the mitochondrial pool of JNK following 6-OHDA 98

4.3.3 Translocation of c-Jun N-terminal kinase 2 (JNK2) to mitochondria 98

4.3.4 Upstream kinases and JNK scaffolds in mitochondrial fractions 102

4.4 Inhibition of JNK and ROS production 104

4.4.1 JNK inhibition does not prevent from oxidative stress 104

4.4.2 Anti-oxidant mediated neuroprotection against ROS generation 105

4.5 Involvement of JNK isoforms in neurite outgrowth 107

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5. DISCUSSION 110

5.1 6-OHDA-induced cell death 110

5.2 6-OHDA toxicity to mitochondria 1125.3 Profound and early oxidative stress in PC12 cells, but not

isolated mitochondria following 6-OHDA 114

5.4 6-OHDA-induced JNK2 activation and translocation to mitochondria

116

5.5 Intracellular JNK pools 1185.6 Upstream and downstream effectors of JNK signaling at the

mitochondria 119

5.7 Time course of pathological events mediated by 6-OHDA 121

5.8 JNK inhibition and protection from oxidative stress 122

5.9 JNK in neuronal death and survival 123

5.10 Technical considerations 125

5.10.1 The PC12 cell model 125

5.10.2 Preparation of isolated mitochondria 125

6. REFERENCES 127

7. ABBREVIATIONS 158

8. DANKSAGUNG 162

9. LEBENSLAUF 163

10. ERKLÄRUNG ZU §10 ABS. 2 NR. 2 DER PROMOTIONS-ORDNUNG

165

11. SUMMARY 166

12. KURZFASSUNG 167

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1. INTRODUCTION

1.1 Parkinson’s disease (PD)

Parkinson’s disease (PD), first described by James Parkinson in 1817 (Parkinson, 1817), is a

progressive neurodegenerative disorder characterized by a preferential loss of dopaminergic

neurons in the substantia nigra pars compacta (Tretiakoff, 1919, for review see Beal, 1995).

This brain structure is essential for the initiation of movement. Consequently, the diagnosis of

PD is usually given by the clinician from the cardinal features of bradykinesia with at least

one or more of the following: resting tremor, gait difficulties, postural instability, and/or

rigidity. Responsiveness to dopamine (DA) replacement treatments is taken as supportive

evidence for the diagnosis. The London Brain Bank criteria for the diagnosis of PD include

neuronal loss in the substantia nigra and presence of Lewy bodies, deposites formed from

fibrillary α-synuclein and hyper-phosphorylated neurofilament protein (Hughes et al., 1992).

More than 90% of PD patients are likely to have Lewy bodies in the substantia nigra in the

end stage of he disease (Hughes et al., 2001). However, there is no evidence that Lewy bodies

would be specific for PD since they are found in a variety of other age-related disorders, and

even in aging brains of people without any signs of locomotor impairment or dementia (Arai

et al., 1992; Forno, 1986). Today, PD is the second most prevalent neurodegenerative disease.

Its occurrence increases with age and is considered to affect more than 2% of the population

beyond 65 years. Less frequently, PD may have an onset below 40 years. This early onset

usually coincides with familial genetic defects (Blum et al. 2001; Golbe et al., 1991; Hardy et

al., 2006).

1.1.1 Current treatment of PD Current conventional treatment is directed to restore voluntary movement by substitution of

dopamine input to the caudate nucleus/putamen and subsequently the motor cortex. The

prodrug levodopa (the natural precursor in dopamine synthesis L-DOPA) is converted to

dopamine by the aromatic L-amino acid decarboxylase (DOPA decarboxylase, DDC). DDC

inhibitors (Carbidopa, Benserazid) do not pass the blood brain barrier, consequently they

reduce peripheral side effects and enhance levodopa efficacy. Inhibitors of the monoamine

oxidase B (MAO-B) – selegiline or rasagiline – raise dopamine levels by blocking its

degradation. A direct effect on dopamine D1 and D2 receptors in the putamen is provided by

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dopamine agonists such as bromocriptine, pergolide or cabergoline. Initially, patients respond

well to this dopamine replacement therapy but treatment is effective for only a limited period

and fails to halt disease progression (Rang et al., 2003).

1.1.2 PD in clinical research

Even though the neurochemical defects and the neuropathological characteristics of PD are

well defined, the etiology of the disease is still unclear. The first clinical signs of PD can only

be observed when the loss of dopaminergic neurons is already strongly advanced (typically

more than 80%), which increases the difficulty to determine the real onset of neuro-

degenerative processes and to identify early changes in the brain. Another drawback are

technical problems, a limited number of reliable, non-invasive imgaging techniques and early

disease markers (Bostantjopoulou et al., 1997; Younes-Mhenni et al., 2007). Additionally, the

progressive mode hardly allows follow-up studies, with only a few neurons dying everyday

(Mochizuki et al., 1996). Data that are obtained from autopsy brains or patients diagnosed

with PD may not at all or not sufficiently reflect events the disease originated from (Hirsch et

al., 1998; Hunot et al., 1997; Tatton et al., 1998).

Smoking, higher coffee and caffeine intake are associated with a significantly lower risk of

PD (Logroscino, 2005; Ross et al., 2000). Infections in early life increase the risk of

progressive dopaminergic cell death. Anti-inflammatory drugs can reduce incidence of PD in

clinical studies, but do not stop the disease progression in patients already diagnosed with PD

(Logroscino et al., 2005). Furthermore, the exposure to pesticides strongly contributes to the

progressive degeneration of dopaminergic neurons (Langston, 1998; Tanner et al., 1999).

This has led to the development of several experimental models reproducing the human

disease, mostly based on the administration of a single neurotoxic compound in vivo or in

vitro.

1.1.3 Experimental PD models

Environmental toxins modelling PD include 6-hydroxydopamine (6-OHDA), 1-methyl-4-

phenyl-1,2,3,6-tetrahydropyridine (MPTP) or the metabolite 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridinium ion (MPP+), rotenone, maneb, and paraquat (Betarbet et al. 2000;

Manning-Bog et al. 2002; McCormack et al. 2002; Sherer et al. 2003; Thiruchelvam et al.

2000). The amount of model toxins gives rise to the crucial involvement of environmental

factors in the pathogenesis of PD (Blum et al., 2001a; Grünblatt et al., 2000; Meco et al.,

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1994; Snyder and D’Amato, 1985). Despite a number of differences between the pathological

symptoms induced by these substances, common mechanisms include mitochondrial

impairment, with great evidence for the inhibition of complex I from the electron transport

chain, and reactive oxygen species (ROS) generation (Ikebe et al., 1995; Kapsa et al., 1996;

Mizuno et al., 1989; Schapira, 1994; Sherer et al. 2002). Environmental PD models are often

not able to reflect relevant features of the disease; e.g. the progressive dopaminergic cell death

is only characteristic following 6-OHDA, and Lewy bodies are only found after rotenone

treatment (Betarbet et al., 2000; Jeon et al., 1995). In this study, 6-OHDA and MPP+ were

used.

1.1.3.1 6-Hydroxydopamine (6-OHDA)

6-Hydroxydopamine (6-OHDA) was the first agent used to produce an animal model of PD

(Ungerstedt, 1971). The toxin is the hydroxylated analogue of the natural neurotransmitter

dopamine. Like dopamine, it is not able to pass the blood brain barrier, so 6-OHDA has been

studied via direct administration to the nigra, the striatum or the medial forebrain bundle

(MFB) of rodent brains or in cell culture models. The bilateral 6-OHDA lesion of the striatum

stands the advantage of being the best suitable model, mimicking closely the human disease

(Deumens, 2002). In low concentrations (≤ 50 µM) 6-OHDA promotes apoptotic cell death,

higher concentrations induce necrotic features as well (Dodel et al., 1999; Walkinshaw and

Waters, 1994).

Biochemical hallmarks of 6-OHDA-mediated cell death are oxidative stress, mitochondrial

dysfunction and activation of intracellular “stress pathways”, namely c-Jun N-terminal/stress-

activated kinases (JNK) and NFĸB-signaling (Blum et al., 2001b; Choi et al., 1999). Yet the

mechanisms, especially the interconnection between these means of action, remain

controversial and poorly understood (Blum et al., 2001a; Jenner, 2003; Walkinshaw and

Waters, 1994).

Oxidative stress is the imbalance between ROS (in particular hydrogen peroxide), generated

by 6-OHDA and dopamine metabolism, and intra- or extracellular autoxidation of 6-OHDA

(Blum et al., 2000; Cohen and Heikkila, 1974; Saner and Thoenen, 1971; Slivka and Cohen,

1985). Autoxidation of 6-OHDA generates p-quinones, toxic intermediates that target

mitochondria and that are eliminated by the conversion to melanine (Arriagada et al., 2004;

Asanuma et al., 2004). During the quinone detoxification process an array of other free

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radical species, such as hydrogen peroxide, superoxide anions, and hydroxyl radicals, are

generated (Blum et al., 2001a). The major source of reactive oxygen species (ROS) are

mitochondria. Oxidation events can be amplified by cytoplasmic free calcium and/or ferrous

iron (Youdim et al., 1990). Increased levels of ROS lead to the damage of cellular

macromolecules and their subsequent peroxidation (Dexter et al., 1989). The importance of

ROS in this model of Parkinson’s disease is fortified by findings that support of the cell-own

antioxidant defense system is highly protective, including administration of glutathione, and,

most effectively, catalase, which scavenges a cell from hydrogen peroxide by decomposition

to water and oxygen (Blum et al. , 2000; Hanrott et al., 2006).

6-OHDA impairs the activity of the mitochondrial complex I of the mitochondrial respiratory

chain, thereby reducing oxidative phosphorylation/ATP production and promoting the

generation of reactive oxygen species (Glinka et al., 1996), yet the effect has not been

demonstrated so clearly as for the other model toxins and remains controversial (Wu et al.,

1996). However, 6-OHDA induces a ROS-related collapse in mitochondrial membrane

potential (Lotharius et al., 1999) and is a strong uncoupler of oxidative phosphorylation

leading to cytochrome c release and further caspase activation (Ochu et al., 1998). 6-OHDA

activates JNK and mediates neuronal cell death (Blum et al., 2001a; Hara et al., 2003).

However, the mechanism of this action is not yet understood.

1.1.3.2 MPTP

In 1982, the neurotoxin MPTP (an analogue of the narcotic meperidine) was discovered

accidentally (Langston et al., 1983). Young drug addicts developed a parkinsonian syndrome

after self-administration of a “synthetic heroin” (MPPP, 1-methyl-4-phenyl-propion-

oxypiperidine). MPTP was identified as the neurotoxic contaminant responsible for this effect

(Ballard et al., 1985; Langston et al., 1983; Langston and Ballard, 1983). MPTP is highly

lipophilic and crosses the blood brain barrier easily. Mono-amine oxidase B (MAO-B) in

astrocytes converts MPTP to the active metabolite MPP+, which is a substrate for the

dopamine transporter (DAT) and accumulates in DA neurons (Javitch et al., 1985). MPP+

inhibits mitochondrial complex I and ultimately leads to cell death (Gluck et al., 1994;

Markey et al., 1984; Tipton et al., 1993). Oxidative stress and the accumulation of free

radicals cause cell death in this model, but ROS are elevated only at very high concentrations

of MPP+ (Chen et al., 2006; Shoffner et al., 1991). However, antioxidant strategies increase

survival also of MPP+-intoxicated neurons (Hantraye et al., 1996; Kitamura et al., 1998; Park

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et al., 2004; Przedborski et al., 1992; Przedborski et al., 1996; Sawada et al., 1996). There is

evidence that the toxicity of MPP+ is mediated via JNK stress kinase signaling (Hunot et al.,

2004; Wang et al.; 2004).

1.3.4 Genetic factors and implications for idiopathic PD

Recently, a number of genes with implications in PD have been discovered (PARK family, for

review see Thomas and Beal., 2007). Mutations in the parkin gene (PARK-2), the PINK1

[phosphatase and tensin (PTEN) homolog-induced putative kinase 1] gene (PARK6), and the

DJ-1 gene (PARK7) lead to a juvenile onset of PD, marked by nigral cell death without Lewy

bodies (Farrer et al., 2001; Miller et al., 2003; Valente et al., 2004). Mutations in the LRRK2

gene (PARK8) lead to classical PD pathology, but the appearance of Lewy bodies is not a

necessary event in the toxicity of the model (Singleton et al., 2005). Mutations in the SNCA

gene (PARK1/4), which encodes for α-synuclein, were associated with a variable onset age of

the locomotor disease (Polymeropoulos et al., 1996).

Gene mutations account only for a minor number of PD cases (about 5%). Some changes do

not result in locomotor impairment, but it has been demonstrated that the genetic background

can enhance toxicity of environmental factors (Zimprich et al., 2004). Genetic models of PD

have shed a new light on mechanisms that could explain the etiology of the disease.

Interestingly, those very recent discoveries about proteins encoded by the PARK gene family

have strengthened again the importance of oxidative stress (DJ-1 as a ROS sensor; Kim et al.,

2005; Park et al., 2005), mitochondrial dysfunction (α-synuclein; Klivenyi et al., 2006; Song

et al., 2004) and stress kinase signaling pathways in PD (parkin, LRRK2, PINK1; Beilina et

al., 2005; Cha et al., 2005; Leutenegger et al., 2006; Li and Beal, 2005).

1.2 Oxidative stress

Molecular oxygen O2 contains two unpaired electrons in its outer orbital and is therefore a

bi-radical. Though relatively unreactive this triplet oxygen can be activated, e.g. by light, to

singlet oxygen with antiparallel electrons and a free π orbital that readily accepts paired

electrons. These molecular forms of oxygen, radicals and other non-radical derivatives of

oxygen can be formed in aerobic organisms, that are equipped with the systems to exploit

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oxygen to their advantage (energy production, cellular defense, enzyme activity, metabolism),

and mechanisms to detoxify the components.

Under physiological conditions the main source for intracellular oxygen radicals is the

electron transport chain of mitochondria (Boveris et al. 1972; Boveris and Chance, 1973;

Loschen et al. 1971). During the flow of electrons along the complexes of the respiratory

chain, leakage of electrons can occur. Those are then transferred directly to molecular oxygen

under formation of the superoxide radical anion O2•-. It is estimated that about 1-3 % of

oxygen consumed in mitochondria are transformed to superoxide under physiological

conditions (Boveris and Chance, 1973). Superoxide anions are highly reactive, but their

damaging effect in cells is limited since diffusion across biological membranes is minimal

due to the negative charge. However, superoxide can be converted to other ROS, among these

are hydrogen peroxide derived from further reduction as well as peroxynitrite formed in a

reaction of superoxide with nitric oxide. Hydrogen peroxide shows limited reactivity but can

readily diffuse across membranes (Loschen et al. 1973). It can be activated by transition metal

ions like ferrous iron to form the highly reactive hydroxyl radical in the Fenton reaction

(Figure 1):

A B (I) Fe2+ + H2O2 → Fe3+ + HO• + OH- (I) 2 H+ + 2 O2

•- → O2 + H2O2

(II) O2•- + Fe3+ → O2 + Fe2+ (II) 2 H2O2 → O2 + 2 H2O

(III) O2•- + H2O2 → O2 + HO• + OH- (III) H2O2 + 2 GSH → GSSG + 2 H2O

(IV) GSSG + NADPH + H+ → 2 GSH + NADP+

Figure 1. ROS generation and scavenging

Chemical reactions facilitating oxygen radical generation (A) and scavenging (B). (AI) Fenton reaction. (AI+II) = (AIII) Haber-Weiss reaction. (BI) Dismutation that can be accelerated by SOD. (BII) Catalase reaction. (BIII) GPx reaction. (BIV) GSH reductase reaction. Abbreviations: GSH (glutathion), GPx (GSH peroxidases), ROS (reactive oxygen species), SOD (superoxide dismutase)

Superoxide radicals can participate in Fenton reactions by reducing Fe3+ to Fe2+ and can thus

propagate reaction (Figure 1, AI) by providing reduced transition metal ions (AII). The net

reaction of (AI) and (AII) is the so-called Haber-Weiss reaction (AIII). Apart from iron, other

transition metal ions, especially copper ions, can participate in the above reactions.

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In general, oxidative stress describes a state of imbalance between the production and

detoxification of reactive oxygen species (Sies, 1991). Therefore, increased production of

ROS as well as impaired antioxidant defense can both contribute to increased accumulation of

ROS. Oxygen radicals can be detrimental to almost any component of cells, including DNA,

proteins and lipids. Cells react upon oxidative stress either by adaptive responses leading to

activation of repair mechanisms or – if the damage is severe – by induction of cell death.

Damage to the electron transport chain or the mitochondrial membrane results in burst of

superoxide production and, consequently, the conversion to hydrogen peroxide and the

hydroxyl radical (Lenaz, 2001; Ueda et al., 2002). The mitochondrial complexes I and III of

the respiratory chain are the most important sites of ROS formation (Boveris et al. 1972;

Turrens and Boveris 1980; Votyakova and Reynolds 2001), complex I being the major and

relevant site of superoxide generation (Liu et al. 2002). ROS themselves can inhibit the

respiratory chain. Hydroxyl radical derived from hydrogen peroxide is the most efficient

inhibitor of complex I among the ROS tested (Zhang et al. 1990).

Although the majority of ROS derives from mitochondrial sources there are numerous other

production sites as well. The enzymes cyclo-oxygenase (COX) and lipoxygenase, involved in

inflammation processes, generate lipid peroxides and oxygen radicals. The lysosomal enzyme

myeloperoxidase catalyzes the production of bacteriocidal hypochlorite from hydrogen

peroxide and chloride. Xanthine oxidase and aldehyde oxidase convert oxygen to superoxide

in the cytosol of endothelial cells. These other sources are important in inflammation and

ischemia/reperfusion injuries (Ferrari et al., 2004; McCord, 1985). Enzymatic oxidation is the

main detoxifying method in the metabolism of xenobiotics, and largely localized to micro-

somes. The main component is the monooxygenase cytochome P450, which is not only

expressed in the liver, but also in catecholaminergic neurons of e.g. the substantia nigra

(Bernhardt et al., 1996). The superoxide radical anion can be generated both from dissociation

of the oxygenated complex or the autoxidation of cytochrome P450 reductase (Denisov et al.,

2007).

The brain is metabolically one of the most active organs. 2% of the total body weight

accounts for 20% of total O2 consumption. Accompanied by that, the human brain faces a

high oxidative burden that can only be alleviated by strong defense mechanisms.

Dopaminergic neurons are even more susceptible, because already the enzymatic (e.g.

MAO-B) and non-enzymatic metabolism of dopamine generates reactive oxygen species.

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Along with the ROS come highly reactive intermediate metabolites. The leukoaminochrome

ο-semiquinone radical that leads to the disruption of the mitochondrial membrane potential

(∆ΨM) and cell death (Arriagada et al., 2004; Asanuma et al., 2004; Izumi et al., 2005).

1.2.1 Antioxidant defense Superoxide anions are first in line in ROS generation, and most other ROS arise directly or

indirectly from superoxide. Therefore, superoxide-detoxifying enzymes act at this early stage

of enzymatic ROS defense (Forman and Azzi, 1997). Superoxide itself can undergo a

dismutation reaction where two molecules of superoxide react to form molecular oxygen and

hydrogen peroxide. This reaction is accelerated by superoxide dismutase (SOD) enzymes

(Figure 1, BI). Two superoxide dismutases have been identified: copper-zinc-dependent

Cu/Zn-SOD (SOD-1) and manganese-dependent Mn-SOD (SOD-2). While Mn-SOD is

mainly localized to mitochondria, Cu/Zn-SOD has been found at high levels in cytosol, but

also in the intermembrane space between the inner and outer mitochondrial membrane

(Okado-Matsumoto and Fridovich, 2001).

Superoxide dismutase reactions result in formation of hydrogen peroxide which has to be

decomposed further. This can be achieved by the reactions of catalase or glutathione

peroxidases (GPx). While catalase activity directly inactivates hydrogen peroxide yielding

water and molecular oxygen (Figure 1, BII), glutathione peroxidases need reduced

glutathione (GSH) as a cosubstrate, oxidizing two molecules to the glutathione disulfide

(GSSG) (Figure 1, BIII). Glutathione peroxidases seem to be in large part responsible for

removal of physiological hydrogen peroxide levels, whereas catalase activity with its high Km

becomes important at abnormally high hydrogen peroxide concentrations (Makino et al.,

1994). However, catalase activity is low in the brain (Marklund et al., 1982). GPx are

distributed in the cytosol as well as the mitochondrial matrix (Vitorica et al., 1984), where

they may act in concert with SODs to decompose mitochondria-derived ROS. In brain tissue,

GPx activity is mainly localized to astroglia (Damier et al., 1993; Takizawa et al., 1994). The

reduced glutathione consumed by GPx reactions is restored from oxidized GSSG by the

glutathione reductase reaction (Figure 1, BIV). Glutathione S-transferases (GST) are

important phase II enzymes in xenobiotic metabolism and conjugate highly reactive

intermediates, drug metabolites or lipid peroxides to GSH (Goon et al., 1993; Hartley et al.,

1995). Another important enzymatic system contributing to antioxidant defense is the

thioredoxin/thioredoxin reductase system. Thioredoxin (Trx) is a polypeptide containing two

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adjacent thiol groups that can regenerate protein disulfide bonds formed under oxidative

conditions. Trx is then regenerated by thioredoxin reductases (Chae et al., 1999).

Low-molecular-mass antioxidants are either produced endogenously, for example glutathione,

uric acid, coenzyme Q, lipoic acid and bilirubin, or they are taken up by the diet. The most

important endogenous antioxidant is the tripeptide glutathione. Glutathione is composed of

the amino acids γ-glutamate, cysteine and glycin. The antioxidant properties of glutathione

are due to the thiol residue in cysteine. Glutathione is found in millimolar concentrations in

most mammalian cells (Cooper and Kristal, 1997). It can react either directly with ROS like

superoxide, nitric oxide or the hydroxyl radical (Winterbourn and Metodiewa, 1994) or act as

cofactor for the enzymatic antioxidant defense by glutathione peroxidases. In brain,

glutathione metabolism seems to be a complex interplay between astrocytes and neurons,

where astrocytes have an essential function in providing neurons with glutathione precursors

(Dringen, 2000).

The most prominent dietary anti-oxidants are vitamin C (ascorbic acid) and vitamin E

(tocopherols). Vitamin C can donate one electron to free radicals under formation of the

ascorbyl radical. Due to mesomeric stabilization of the free electron in the ascorbyl radical,

this component is very stable. Lipid-soluble tocopherols are more effective against radicals

generated in membranes (e.g. mitochondria). However, to date there is no evidence that levels

of vitamines are altered in PD patients.

There are many phytochemicals that exhibit antioxidant properties, including carotenoids,

flavonoids and anthocyanidines. Their efficacy might be particulately higher than classical

antioxidants. Their lipophilic character may allow easy penetration of the blood brain barrier.

Furthermore, several compounds were found to activate transcription factors for

cytoprotective genes, e.g. NF-E2-related factor-2 (Nrf-2). Among those substances are the the

phytoalexin resveratrol (Hsieh et al., 1999; Manna et al., 2000), and flavonoids, e.g. quercetin

(Hanneken et al., 2006). Flavanols are therefore highly potential antioxidants (Figure 2).

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Figure 2. Antioxidant potential of flavonoids

Structure of the flavonol quercetin showing features important in defining the classical anti-oxidant potential of flavonoids. The most important of these is the catechol or dihydroxylated B-ring (shaded yellow) that easily donates a proton to stabilise a radical species. Other important features include the presence of unsaturation in the C-ring (shaded red) and the presence of a 4-oxo function in the C-ring (shaded green), increasing the potential to delocalize one electron. The catechol group and other functions may also ascribe an ability to chelate transition metal ions such as copper and iron (shaded blue). Adapted from Spencer et al. (2003), Biochemical Journal, 372, 173–181.

1.2.2 Role of ROS and ROS products in cell death

Excessively elevated intracellular ROS levels lead to lipid peroxidation, oxidation of proteins,

and DNA damage. Unsaturated fatty acids are an easy target for ROS. Oxidated lipids lead to

membrane dysfunction, while the products of lipid decomposition, malondialdehyde and

4-hydroxynonenal, can exert cytotoxic actions, including DNA and mitochondrial damage

(Eckl et al., 1993; Benamira et al., 1995; Keller and Mattson, 1998, Siems et al., 1996; Lu et

al., 2002).

Oxidative protein modifications include a wide variety of reactions depending on the type of

amino acids that are affected: sulfur-containing moieties in cysteine and methionine are easily

oxidized to disulfides or sulfoxides, respectively, basic amino acids arginine and lysine can be

oxidized to aldehydes, aromatic rings in amino acids can be oxidized or nitrated and aliphatic

carbon atoms can be oxidized to alcohols. Apart from direct attack of ROS, proteins can also

be oxidatively modified by lipid peroxidation products or by sugars and aldehydes that lead to

INTRODUCTION

19

formation of advanced glycation end products (Münch et al., 1997). Repair mechanisms

include restoration of cysteine thiol groups from disulfides, e.g. by thioredoxin, and

restoration of methionine from sulfoxides by methionine sulfoxide reductase (Stadtman,

2004). However, oxidized proteins can be removed by proteolysis via the proteasome

complex.

Increased levels of ROS can either induce adaptive responses or elicit cell death. Often, mild

oxidative stress leads to activation of antioxidant responses and resistance to higher ROS

levels. Intracellular signaling triggered by ROS is very complex (reviewed in Finkel, 1998;

Dalton et al., 1999; Allen and Tresini, 2000). As a result, several transcription factors can be

affected by ROS (reviewed in Sun and Oberley, 1996; Sen and Packer, 1996; Dalton et al.,

1999). One of these signaling cascades is the mitogen-activated protein kinase (MAPK)

pathway (see section 1.5), which activates the transcription factor AP-1. AP-1 is a dimer of

two proteins, c-Fos and c-Jun, existing either as an inactive homodimer of phosphorylated c-

Jun or an active heterodimer of c-Jun and c-Fos. A consensus sequence for AP-1 binding has

been found in the antioxidant response element (ARE). Among the genes with ARE

sequences are glutathione-S-transferase and glutathione synthesizing enzymes, NAD(P)H

quinone oxidoreductase-1, ferritin, heme oxygenase-1 and phase II detoxification enzymes

(La Fauci et al., 1989; Trejo et al., 1994). The primary transcription factor controlling ARE is

Nrf-2 (Chen et al., 2004). Nrf-2 binds to ARE by homo- or heterodimerization with other

leucine-zipper transcription factors, e.g. c-Jun (McMahon et al., 2001; Venugopal and

Jaiswal, 1998; Xu et al., 2006). Thereby, Nrf-2 can protect from mitochondrial toxins used as

models for PD (Lee et al., 2003).

Damage of cellular components by ROS and insufficient repair mechanisms usually lead to a

cell death. Whether cells die by apoptosis or necrosis is largely dependent on the severity of

the insult: apoptosis, which is an energy-consuming process, requires some residual

functionality of cellular proteins and ATP (Richter et al., 1996), whereas severe oxidative

damage with destruction of cellular integrity and dissipation of ATP levels usually elicits

necrotic cell death (Leist et al., 1997; Eguchi et al., 1997).

INTRODUCTION

20

1.3 Hallmarks of apoptosis

Apoptotic cell death can be initiated by various other (external and internal) signals apart

from ROS and executed by several interrelated pathways. Cells remain intact and do not

cause an inflammatory response in situ. However, there are clear and specific signs for

apoptotic events: cell shrinkage, membrane budding, chromatin aggregation, margination of

condensed chromatin at nuclear membrane, DNA-laddering in agarose gel, and the formation

of apoptotic bodies (fragments of condensed nucleus surrounded by small rim cytoplasm).

The process is energy-dependent and well-controlled, apoptotic cells and bodies are cleared

promptly from the tissue by phagocytosis (macrophages or glial cells).

At the molecular level, the apoptotic cell death machinery forms a complex cascade of

ordered events, controlled by the regulated expression of apoptosis-associated genes and

proteins. The apoptotic pathways have been investigated intensely, with the attempt to

catagorize them. As the number of stimuli and pathways is great so are the categories of

programmed cell death by now: intrinsic and extrinsic, mitochondrial and death receptor, p53-

dependent and -independent, caspase-dependent and -independent. It is apparent that

apoptosis is not a series of clearly defined pathways but a complex network of signaling

systems, that, if certain mechanisms fail, can also escape to other forms of cell death,

autophagy or necrosis (Lockshin and Zakeri, 2007). Complicating the attempt of

pharmacological intervention of these pathways, apoptosis is an essential component of

neuronal plasticity (Yousefi et al., 2003). Cell death in animals is normally classified as type I

(apoptotic), type II (autophagic) or type III/necrotic (Penaloza et al., 2006). Signaling

networks converge at the level of caspase cascades. Caspases, a family of cysteine proteases,

are central mediators of apoptosis. Executioner caspases cleave essential cellular proteins.

Their activation in 6-OHDA toxicity has been described previously (Ochu et al., 1998;

Woodgate et al., 1999).

1.4 Mitochondria in neuronal cell death

Mitochondria make up to 40% of the cell volume in cells that are most metabolically active:

muscle cells and neurons. A mitochondrion has two membranes: The outer mitochondrial

membrane (OMM) contains small pores, freely permeable to ions and other small molecules.

INTRODUCTION

21

The inner membrane is highly impermeable, even to protons. The proton gradient across the

inner membrane creates an electric potential and is used to generate ATP molecules. This is

preceded by the consecutive transfer of electrons on molecular oxygen by four protein

complexes attached to the inner wall of the inner mitochondrial membrane (IMM). These

protein complexes are identified as Complex I, II, III and IV and comprise the respiratory

chain ("electron transport chain") (for review see Gray et al., 1999). High mitochondrial

activity entails a low percentage of an incomplete transfer of electrons on molecular oxygen

resulting in ROS (Boveris et al., 1976; Turrens and Boveris, 1980). During apoptosis

mitochondria are a target of signaling molecules that alter the permeability of the

mitochondrial membranes. This can lead to disturbance in mitochondrial energy production

and to an increase in ROS, as well as to the disruption of the mitochondrial membrane

integrity, loss of the mitochondrial membrane potential (∆ΨM), swelling and release of pro-

apoptotic molecules (Bernardi et al., 1998; Halestrap et al., 1998; Kroemer et al., 1998;

Crompton, 1999). Therefore, mitochondria play a central role in cell death signaling pathways

(for reviews see Desagher and Martinou, 2000; Green and Reed, 1998; Kroemer et al., 1998;

Susin et al., 1998).

Among the molecules that are released from the compartment between the IMM and the

OMM, the intermembrane space (IMS), during apoptosis are apoptosis-inducing factor (AIF),

endonuclease g (EndoG), second mitochondria-derived activator of caspases/direct IAP-

binding protein of low isoelectric point (smac/DIABLO), HtrA2/Omi, and cytochrome c

(cyt c). AIF and EndoG translocate to the nucleus and induce large-scale DNA fragmentation,

thus leading to chromatin condensation (Daugas et al., 2000). Smac/DIABLO and HtrA2/Omi

potentiate caspase-dependent cell death by neutralizing the effect of proteins that function as

inhibitors of apoptosis (Rehm et al., 2003; Verhagen et al., 2000). Their involvement in the

apoptotic machinery is preceded by the release of cyt c, which is a key molecule in the

activation of caspases (Liu et al., 1996; Andreyev et al., 1998; Kantrow and Piantadosi, 1997;

Susin et al., 1999; Zamzami et al., 1997; Kluck et al., 1997; for review see Kroemer and

Reed, 2000; Polster and Fiskum, 2004).

Cyt c participates in the electron transport chain by passing electrons from complex III to

complex IV. The IMS contains 15% free cyt c while the rest is situated in the cristae to exert

the function described above (Bernardi and Azzone, 1981). In the IMS cyt c can act as an

antioxidant, accepting an electron from the superoxide anion (Skulachev, 1998). In apoptotic

conditions the OMM becomes permeable for larger molecules, resulting in a release of cyt c

INTRODUCTION

22

into the cytosol. Cyt c forms a complex with apoptosis protease activating factor 1 (APAF-1),

pro-caspase 9 and ATP, which in turn activates caspases 3/7 (Li et al., 1997; Zou et al.,

1999). These executioners of apoptosis cleave a variety of essential proteins and activate other

proteases and DNAses resulting in cell death (Leist and Jäättelä, 2001).

Transport of larger substances across mitochondrial membranes requires the opening of

transition pores (Zamzami et al., 1996, Scarlett and Murphy, 1997). The mitochondrial

permeability transition pore (mPTP) is a complex of the voltage dependent anion channel

(VDAC) and bax protein (Shimizu, 1999; Blatt and Glick, 2001). It is a water-filled pore,

through which molecules up to 1500 D (IMM) or even higher (OMM) can leak in or out (e.g.

cytochrome c). At low-level conductance the PTP opening is reversible, causing a collapse of

the mitochondrial membrane potential (Ichas and Mazat, 1998). ATP can still be generated

due to the activity of an external respiratory chain, a complex in the OMM (Skulachev, 1998).

At high-level conductance the PTP opening is irreversible and leads to swelling of the

mitochondrial matrix and the loss of pyridine nucleotides and substrates for the citric acid

cycle in the mitochondrial matrix (Kantrow and Piantadosi, 1997; Susin et al., 1998;

Zamzami et al. 1997; Kluck et al., 1997). Upon a stimulus mitochondria react individually

within a cell, so that, to a certain extent, impaired mitochondrial function can be compensated

(Collins et al., 2002).

The mechanism of pore formation/opening is regulated by members of the Bcl-2 family.

These include the anti-apoptotic Bcl-2 and Bcl-XL, which reside on the OMM, and the

proapoptotic Bax and Bak, which are predominantly cytosolic while they can also be found

connected to the surface of the OMM (Zimmermann et al., 2001). During apoptosis, Bax is

activated, translocates to mitochondria forming pores of various diameters in the OMM that

can be large enough to release cyt c. On the other hand, anti-apoptotic Bcl-2 can inhibit cyt c

release to the cytosol, and prevent Bax activation, thereby protecting cells from death (Kluck

et al., 1997; Yang et al., 1997). BH3 domain-only Bcl-2 family members (e.g. Bid, Bim, Bad)

promote pro-apoptotic effects indirectly by inhibiting binding of Bcl-2 to Bax, thus freeing

Bax to be incorporated into the mitochondrial membrane (Gross et al., 1999; Zong et al.,

2001). The JNK stress kinases can activate bax proteins, targeting bax to the OMM (Tsuruta

et al., 2004).

INTRODUCTION

23

Increased ROS, in particular superoxide, can induce the formation of the mPTP due to

disulfide cross-linking (Kowaltowski et al., 1998; Zamzami et al., 1998). However, it is not

clear whether an increased ROS production precedes the mPTP opening or if the rise in ROS

production is the result of the permeability change of the mitochondrial membranes.

Oxidative stress and mitochondrial dysfunction are implicated to play a major role in

PD-related neurodegeneration (Greenamyre et al., 1999; Jenner and Olanow, 1998; Schapira,

2008). A significant and specific reduction in the activity and amount of complex I was found

in PD patients (Ramsay et al., 1989; Schapira et al., 1990; Parker et al., 1989; Mizuno et al.,

1989). The inhibition of complex I results in ROS formation, in a mechanism independent of

the ∆ΨM (Sipos et al., 2003). Additionally, complex I is highly susceptible to an oxidative

attack itself (Zhang et al. 1990). Environmental toxins relevant in PD target complex I to

various extents (see section 1.1.3.1). However, a combined action of respiratory chain

inhibition and additional oxidative stress is needed to induce apoptosis (Chinopoulos and

Adam-Vizi, 2001). Even up to date there is a controversy as to the point at which oxidative

stress and mitochondrial dysfunction in PD first occur (Jenner and Olanow, 2006). Is there a

common signaling pathway that would control both, mediate signals from ROS to

mitochondria or the other way around?

1.5 MAPK cascades

The mitogen-activated protein kinase (MAPK) pathways execute signal transduction from the

cell surface into the nucleus to control gene expression by means of activating transcription

factors. These pathways produce a wide range of cell responses, including cell proliferation,

differentiation and apoptosis (Bonni et al., 1999; Chang and Karin, 2001; Pearson et al.,

2001; Yang et al., 2005).

MAP kinase subfamilies are currently classified in 6 groups: ERK1/2, JNK, p38 MAPK,

ERK5, and the atypical classes ERK3/4 and ERK7/8. The ERKs (the classical ERK1/2,

extracellular signal-regulated kinases) are preferentially activated in response to growth

factors, and regulate cell proliferation and cell differentiation. JNKs (c-Jun N-terminal

kinases, also known as stress-activated protein kinases) and p38 isoforms are responsive to

stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and

INTRODUCTION

24

are involved in cell differentiation and apoptosis. All these pathways contain redox-sensitive

sites (Sen and Packer, 1996). Although exceptions are known, antioxidant compounds

inducing ERK isoforms often elicit cytoprotective signaling, whereas oxidative stress

activates JNK and p38 pathways that mostly result in cell death signaling (Kamata and Hirata,

1999; Poli et al., 2004; Torres, 2003). MAP kinase pathways lead to phosphorylation of AP-1,

c-Jun and c-Fos, thereby affecting gene transcription.

1.5.1 c-Jun N-terminal kinases (JNK)

Three genes, jnk 1-3, encode the JNK family with multiple splice variants (Barr and

Bogoyevitch, 2001). Each of the three JNK isoforms resolves on a SDS-PAGE gel as either

an approximate 46 or 55 kDa protein. JNKs are phosphorylated and activated by the dual

specificity MAPKKs, MKK4 and MKK7 (Figure 3), which phosphorylate JNKs on Thr183

and Tyr185 residues (Kyriakis and Avruch, 2001; Paul et al., 1997). The MAPKKKs, which

in turn activate these two MAPKKs, include ASK1, MEKKs and MLKs (Kyriakis and

Avruch, 2001).

In addition to upstream kinases, scaffold proteins from the JIP family modulate JNK signaling

(Figure 3). They form complexes with JNKs and selected members of the upstream

phosphorylating kinases (Dickens et al., 1997; Whitmarsh et al., 1998). JIPs also likely play a

role to direct JNK and their upstream kinases to different compartments of the cell by

associating to microtubules (Goldstein, 2001; Verhey et al., 2001).

The best analyzed target of JNKs is the transcription factor c-Jun. The activation of c-Jun

requires translocation of activated JNK to the nucleus, and subsequent phosphorylation of two

sites, serine 63 and 73. Then, c-Jun becomes competent to form homo- or heterodimers, or to

act as an AP-1 transcription factor component (Behrens et al., 1999; Hibi et al., 1993;

Dérijard et al., 1994; Kyriakis et al., 1994). Via the pathway JNK – c-Jun apoptotic processes

are regulated (Xia et al., 1995; Kang et al., 1998; Maroney et al., 1998; Le-Niculescu et al.,

1999; Troy et al., 2001; Schlingensiepen et al., 1994). Beyond c-Jun, JNKs also

phosphorylate other transcription factors, including ATF-2 (Gupta et al., 1995), Elk-1 (Yang

et al., 1998) and NFAT (Chow et al., 1997). The three JNK isoforms display differing

affinities and specificity for target transcription factors. JNK2 has a 25-fold greater binding

affinity for the transcription factor c-Jun, which correlates to a 10-fold increase of

phosphorylated c-Jun relative to JNK1 (Kallunki et al., 1994, Sluss et al., 1994). JNK3

INTRODUCTION

25

demonstrates weakest binding to c-Jun (Gupta et al., 1996). There are several other contrasts

between the isoforms. JNK1 and 2 exhibit a broad tissue distribution whereas JNK3 is

restricted to brain and testes (Carboni et al., 1997). In the adult rodent brain, JNK2 and JNK3

are widely expressed, while JNK1 is localized only to some specific regions like hippocampus

(Carboni et al., 1998). Moreover, not only the induction of JNK activity in these tissues per se

but the subcellular localization and access to different substrates is important for specific JNK

functions (Coffey et al., 2000). This is still a matter of intense research. Knock-out mice that

individually lack jnk1, jnk2 or jnk3 genes develop normally (Kuan et al., 1999; Yang et al.,

1997). However, mice deficient in both JNK1 and 2 die prematurely and display brain

abnormalitites that are attributable to a dysregulation of apoptosis (Kuan et al., 1999). This

indicates overlapping regulating and/or compensating abilities of the isoforms.

Figure 3. JNK signaling pathway

Mechanism of c-Jun N-terminal kinase (JNK) signaling by step-wise phosphorylation via upstream kinases. A stimulus mediates activation of members of the MAP3K family (MEKK1 or MLKs), which phosphorylate MAP2K (MKK4 or MKK7). In turn, MAP2Ks activate JNK (A). For activation of a downstream kinase all partners need to be in close proximity which is guaranteed by the scaffold protein JIP (B). Adapted from Chang and Karin (2001), Nature, 410, 37–40. Abbreviations: HPK (hematopoietic protein kinase), JIP-1 (JNK interacting protein-1), JNK (c-Jun N-terminal kinase), MAP2K or 3K (mitogen-activated protein 2 or 3-kinase), MEKK-1 (mitogen-activated protein 3 kinase-1), MKK-4/7 (mitogen-activated protein 2 kinase-4/7), MLK (mixed lineage kinase), P (active phosphate).

A B

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26

JNKs are responsive to stress stimuli, such as cytokines, environmental toxins, oxidative

stress, ultraviolet irradiation, heat shock, and osmotic shock. In certain conditions, JNKs

mediate cell differentiation, but upon a strong stimulus the cell usually undergoes apoptosis

that can be prevented by JNK inhibition (Waetzig and Herdegen, 2004). The endogenous

level of JNK activity, at least in the rodent brain and in some cultured neuron populations, has

been noted to be high (Coffey et al, 2000; Xu et al., 1997). This basal tone of JNK activity is

required for physiological events that sustain normal brain function (Waetzig and Herdegen,

2004).

JNKs play an important role in PD models. 6-OHDA and MPTP (MPP+) are strong activators

of JNK and mediate neuronal cell death (Blum et al., 2001a; Saporito et al., 2000; Bozyczko-

Coyne et al., 2002 Hara et al., 2003; Pan et al., 2007). Consequently, JNK inhibition can

rescue dopaminergic neurons from apoptosis (Saporito et al., 1999; Wang et al., 2004; Cha et

al., 2005; Pan et al., 2007; Chen et al., 2008). Morever, the brains of PD patients display

decreased levels of glutathione, an endogenous suppressor of JNK activity (Wilhelm et al.,

1997; Adler et al., 1999).

JNKs are able to phosphorylate other proteins distant from the common MAPK pathway.

Among such substrates have been identified tumor suppressor p53 (Buschmann et al., 2001),

tau protein (Goedert et al., 1997) and amyloid precursor protein (Standen et al., 2001). JNKs

also interact with the proteins of the bcl-2 family, such as Bcl-2, Bcl-XL, Bad, Bax or Bim,

thereby regulating the mitochondrial death pathway (Park et al., 1997; Kharbanda et al.,

2000; Donovan et al., 2002; Schroeter et al., 2003; Putcha et al., 2003; Tsuruta et al., 2004).

Due to the localization of most BH3-proteins, the authors have pointed out, that JNK must be

in the vicinity of mitochondria (Lei et al., 2002; Aoki et al., 2002). The crucial involvement

of JNKs in the mitochondrial death signaling is supported by the fact that cytochrome c

release into the cytosol can be prevented by JNK inhibition (Tournier et al., 2000; Hatai et al.,

2000; Ichijo et al., 1997). Moreover, JNKs release inhibitors of anti-apoptotic proteins, such

as Smac/DIABLO from the mitochondria (Chauhan et al., 2003).

There are several hints that JNK stresskinases are involved in the mitochondrial pathology

and in ROS generation following 6-OHDA. How these events are connected has not yet been

identified. And would JNKs exert any isoform specificity in compartmental signaling, e.g. at

mitochondria?

AIMS OF THE THESIS

27

2. AIMS OF THE THESIS

Parkinson’s disease (PD) is a progressive neurodegenerative disorder of less defined etiology

and limited treatment options. An important role in the development of the disease has been

addressed to oxidative stress, mitochondrial impairment and stress kinase signaling. How

these key events are initiated and interact with each other, remains unclear. In this thesis, the

well-established 6-OHDA administration to PC12 cells was used as a model of PD. PC12

cells origin from the catecholaminergic rat pheochromocytoma. The present experiments

addressed the following issues:

The establishment of a flow cytometrical detection method for quantification of apoptosis

and necrosis and analysis of PC12 cell death induced by 6-OHDA.

The measurement of reactive oxygen species (ROS) over time to detect onset of and

changes in oxidative stress following 6-OHDA. Determination of the source of ROS.

The setup of a flow cytometrical assay and spectrofluorometrical analyses to follow-up the

mitochondrial membrane potential during 6-OHDA treatment to denote any disturbances and

the final disruption of ∆ΨM.

The analysis of the above mentioned parameters in isolated mice brain mitochondria to

detect similarities and differences in the sequence of pathological events.

The JNK isoform-specific investigation of PC12 mitochondrial fractions, the amount as

well as activation status, and upstream and downstream signaling following 6-OHDA.

The microscopical characterization of the intracellular distribution of JNK isoforms.

The effects of JNK inhibition by SP600125 and the antioxidants methysticin, luteolin,

resveratrol and tert-butylhydroquinone on 6-OHDA-induced oxidative stress.

The involvement of JNK in a model of neurite outgrowth.

MATERIALS AND METHODS

28

3. MATERIALS AND METHODS

3.1 Materials

Unless otherwise indicated, all solutions and dilutions were prepared in double-destilled water

(DDW). All chemicals were pro analysis (p.a.). Apart from antibodies Table 1 gives all

materials used in this study.

Table 1. Materials

Material Manufacturer / Supplier

Acrylamide / bis-acrylamide solution 29:1 Bio-Rad Laboratories, München, Germany

Adenosin-diphosphate Sigma-Aldrich, München, Germany

Agarose SeaKem LE Biozym, Oldendorf, Germany

Albumin, bovine fraction Sigma-Aldrich, München, Germany

6-Aminocaproic acid Merck-Schuchardt, Hohenbrunn, Germany

Ammonium persulphate Merck, Darmstadt, Germany

Annexin V-FITC Sigma-Aldrich, München, Germany

B-27 Supplement Invitrogen, Karlsruhe, Germany

Boric acid Sigma-Aldrich, München, Germany

Bromophenol blue Merck, Darmstadt, Germany

t-Butylhydroquinone Axxora, Lörrach, Germany

Calcium chloride Merck, Darmstadt, Germany

Cell culture dishes (35 mm) Sarstedt, Nümbrecht, Germany

Cell culture plates (10 cm, 6 wells, 24 wells) Nunc, Wiesbaden, Germany

Chamber slides (2 wells) Nunc, Wiesbaden, Germany

Cover glasses Nunc, Wiesbaden, Germany

Cryotubes (2 ml) Greiner Bio-One, Frickenhausen, Germany

Cytosine-ß-D-Arabinofuranoside Sigma-Aldrich, München, Germany

DAB tablets Sigma-Aldrich, München, Germany

2',7'-Dichloro-dihydrofluorescein Sigma-Aldrich, München, Germany


29

2',7'-Dichloro-dihydrofluorescein-diacetate Sigma-Aldrich, München, Germany

Dihydrorhodamine 123 Sigma-Aldrich, München, Germany

Dimethylsulfoxide (DMSO) Merck, Darmstadt, Germany

Dinitrophenol Merck, Darmstadt, Germany

dNTP set (10 mM solutions) Invitrogen, Karlsruhe, Germany

DTT Invitrogen, Karlsruhe, Germany

Dye Reagent Bio-Rad Laboratories, München, Germany

ECL Plus GE Healthcare, Munich, Germany

EDTA Merck, Darmstadt, Germany

EGTA Merck, Darmstadt, Germany

Ethanol p. a. Merck, Darmstadt, Germany

Ethanol, technical (denatured) Bundesmonopol für Branntwein (BfB), Offenbach, Germany

Ethidium bromide solution (10 mg/ml) Invitrogen, Karlsruhe, Germany

Fetal calf serum (FCS) Bio Whittaker, Vervriers, Belgium

Ficoll Miltenyi Biotec, Bergisch-Gladbach, Germany

Filter paper Whatman, Maidstone, UK

Filter unit 0.22 µm, syringe-driven Qualilab, Bruchsal, Germany

Gentamycin Invitrogen, Karlsruhe, Germany

G 418, sulfate (solution) Stratagene, Amsterdam, The Netherlands

D-Glucose Merck, Darmstadt, Germany

Glutamax Invitrogen, Karlsruhe, Germany

Glycerol Merck, Darmstadt, Germany

Glycine Merck, Darmstadt, Germany

HEPES Merck, Darmstadt, Germany

Hoechst 33258 Sigma-Aldrich, München, Germany

Horse serum Invitrogen, Karlsruhe, Germany

6-hydroxydopamine hydrochloride Sigma-Aldrich, München, Germany

Hyperfilm ECL GE Healthcare, Munich, Germany

Immobilon P 1500 Millipore, Schwalbach, Germany


30

Insulin Sigma-Aldrich, München, Germany

JC-1 Sigma-Aldrich, München, Germany

Kaiser’s glycerol gelatine Merck, Darmstadt, Germany

Luteolin Axxora, Lörrach, Germany

Magnesium chloride (PCR) Invitrogen, Karlsruhe, Germany

Magnesium chloride Merck, Darmstadt, Germany

Magnesium sulfate Sigma-Aldrich, München, Germany

Mannitol Merck, Darmstadt, Germany

2-Mercaptoethanol Sigma-Aldrich, München, Germany

Methanol Merck, Darmstadt, Germany

1-Methyl-4-phenylpyridinium iodide Sigma-Aldrich, München, Germany

Methysticin Axxora, Lörrach, Germany

Minimum essential medium (MEM) Sigma-Aldrich, München, Germany

Mitotracker Red CM-H2Xros Invitrogen, Karlsruhe, Germany

Non-fat dry milk Uelzena, Uelzen, Germany

Paraformaldehyde Merck, Darmstadt, Germany

PBS (w/o Ca2+ and Mg2+) Invitrogen, Karlsruhe, Germany

PCR buffer (10 x) Invitrogen, Karlsruhe, Germany

Penicillin/Streptomycin solution (10,000 IU / 10,000 µg/ml)

Invitrogen, Karlsruhe, Germany

Phenylmethylsulfonylfluorid Sigma-Aldrich, München, Germany

Phosphatase Inhibitor Cocktail II Sigma-Aldrich, München, Germany

Pipettes (serological, sterile; 5 / 10 / 25 ml) Sarstedt, Nümbrecht, Germany

Pipette tips (10 / 200 / 1,000 µl) Sarstedt, Nümbrecht, Germany

Poly-l-Lysine Sigma-Aldrich, München, Germany

Ponceau S Sigma-Aldrich, München, Germany

Potassium chloride Merck, Darmstadt, Germany

Potassium dihydrogen phosphate Merck, Darmstadt, Germany

Propidium iodide Sigma-Aldrich, München, Germany

Protease Inhibitor (Complete) Roche Diagnostics, Mannheim, Germany

Protein marker, prestained, broad range New England Biolabs, Frankfurt, Germany


31

Proteinase K Sigma-Aldrich, München, Germany

QIAamp DNA mini kit Qiagen, Hilden, Germany

RPMI-1640 medium Invitrogen, Karlsruhe, Germany

Rotenone Sigma-Aldrich, München, Germany

SlowFade light antifade kit Invitrogen, Karlsruhe, Germany

Sodium bicabonate Sigma-Aldrich, München, Germany

Sodium carbonate Sigma-Aldrich, München, Germany

Sodium chloride Merck, Darmstadt, Germany

Sodium dodecyl sulfate Merck, Darmstadt, Germany

Sodium hydroxide Merck, Darmstadt, Germany

Sodium pyruvate Sigma-Aldrich, München, Germany

Sodium succinate Sigma-Aldrich, München, Germany

SP600125 Axxora, Lörrach, Germany

Staurosporine Axxora, Lörrach, Germany

Taq DNA polymerase Invitrogen, Karlsruhe, Germany

TEMED Carl Roth, Karlsruhe, Germany

Thermanox coverslips Nunc, Wiesbaden, Germany

Transferrin Merck, Darmstadt, Germany

Tris Merck, Darmstadt, Germany

Triton X-100 Merck, Darmstadt, Germany

Trypan blue solution, cell culture tested Sigma-Aldrich, München, Germany

Trypsin Sigma-Aldrich, München, Germany

Trypsin inhibitor Sigma-Aldrich, München, Germany

Tubes (0.5 / 1.5 / 2.0 ml) Sarstedt, Nümbrecht, Germany

Tubes for PCR Sarstedt, Nümbrecht, Germany

Tubes, sterile (15 / 50ml) Sarstedt, Nümbrecht, Germany

Tween-20 Merck, Darmstadt, Germany

Ultrapure water Biochrom, Berlin, Germany

Valinomycin Sigma-Aldrich, München, Germany

Vectastain Elite ABC Vector Labs, Burlingame, USA


32

3.2 Laboratory Equipment

Table 2. Equipment

Equipment Manufacturer / Supplier

Agarose gel electrohoresis Bio-Rad Laboratories, München, Germany

AnalySIS software Soft Imaging System, Münster, Germany

Autoclave DS 202 Webeco, Bad Schwartu, Germany

Cell Quest Pro software BD, Franklin Lakes, USA

Centrifuge; Biofuge fresco, Labofuge GL Heraeus, Osterode, Germany

Centrifuge; Mikrofuge Neolab, Heidelberg, Germany

Cell incubator Heraeus, Osterode, Germany

DMR microscope Leica Microsystems, Wetzlar, Germany

Electrophoresis power supply Invitrogen, Karlsruhe, Germany

FACSCalibur flow cytometer BD, Franklin Lakes, USA

Film processor AGFA, Mortsel, Belgium

Fluoroskan Ascent FL microplate reader Thermo Electron Corporation, Dreieich, Germany

GraphPad Prism Software GraphPad Software, San Diego, USA

Hamilton syringes (10- and 50-µl) Hamilton, Bonaduz, Switzerland

Heating block (Thermomixer 543) Eppendorf, Hamburg, Germany

Incubator (Innova 4000) New Brunswick Scientific, Amsterdam, The Netherlands

Laminar flow unit Heraeus, Osterode, Germany

Leica Qwin software Leica Microsystems, Wetzlar, Germany

Leica DM L microscope with a fluorescence unit (100 W Hg + filter cubes)

Leica Microsystems, Wetzlar, Germany

Microplate reader 680 Bio-Rad Laboratories, München, Germany

MiniProtean II Vertical PAGE chamber Bio-Rad Laboratories, München, Germany

Olympus CK 2 microscope Olympus, Hamburg, Germany

Oxygraph Hansatech Instruments, Norfolk, UK

pH meter WTW, Weilheim, Germany

Rotator (Polymax 2040) Heidolph, Kehlheim, Germany


33

Semi-dry transfer unit (Pegasus) Phase, Lübeck, Germany

Sonicator (Sonopuls GM 70) Bandelin, Berlin, Germany

Spectrophotometer (U-2000) Hitachi, Wiesbaden, Germany

SPSS 14 for Windows SPSS Inc., Chicago, USA

Thermocycler (Personal Cycler) Biometra, Göttingen, Germany

UV light (Image Master VDS) Bio-Rad Laboratories, München, Germany

Water bath Heraeus, Osterode, Germany

WinMDI Software (Ver. 2.8 #13) Freeware, Author: Joe Trotter

Zeiss LSM 5 image browser Carl Zeiss Jena, Jena, Germany

Zeiss LSM 510 laser scanning microscope Carl Zeiss Jena, Jena, Germany

3.3 Methods

3.3.1 PC12 cell culture

The rat pheochromocytoma cell line PC12 was purchased from the German Collection of

Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und

Zellkulturen GmBH; ACC159). The cells were grown in RPMI 1640 medium, supplemented

as described in Table 3. The penicillin/streptomycin solution, horse serum and FCS were

stored at -20° C, and the RPMI-1640 medium was stored in a cooling chamber (4° C). For

maintenance and most experiments PC12 cells were grown in 10 cm culture dishes, for

mitochondrial isolation 20 cm plates were used for better handling of the greater amount of

cells, that were needed to obtain a sufficient yield of isolated mitochondria. The culture

medium was stored at 4° C, and prewarmed to 37° C before giving to the cells. The volume

for 10 cm plates was 7 ml, and 20 ml for 20 cm dishes. At least two times a week about 80%

of the volume was replaced by fresh medium.

PC12 cells are poorly adherent, therefore all plates have to be coated with collagen

(0.1 mg/ml in PBS) in advance. The bottom of the cell culture plates was covered with a thin

layer of collagen solution, placed in the incubator for a minimum of 1 h and then washed with

PBS to remove collagen that had not adhered to the surface. Air-dried plates were stored

under sterile conditions.


34

Table 3. Composition of the PC12 cell culture medium

Cell culture medium

RPMI-1640 medium

Horse serum (HS, inactivated at 56° C for 30 min) 10%

Fetal calf serum (FCS, inactivated at 56° C for 30 min) 5%

HEPES 1%

Sodium pyruvate 1%

Penicillin/Streptomycin 1%

Adjusted with sterile 2 M glucose solution to 4,5 g/l glucose in the culture medium

3.3.1.1 Splitting

The doubling time for PC12 cells is about 50 - 60 h. After approximately one week incubation

cells reached 70 - 80% confluence and were seeded onto new plates (division of 1:3 - 1:6).

For splitting, PC12 cells were pretreated with 0.5 mM EDTA in PBS to reduce cell adhesion.

The following procedure was applied:

1. Cells were washed with PBS (37° C)

2. Cells were rinsed with 2 ml 0.5 mM EDTA in PBS (37° C) and placed for 2 min in the

incubator (37° C, 5% CO2)

3. 5 ml of medium was added (37° C)

4. Cells were scraped off the cell culture plate and transferred into a 15 ml tube and

centrifuged for 10 min; (1000 x g; RT)

5. Pellets were first resuspended in 2 - 3 ml medium, sheared 3x through a 21 G needle/

5 ml syringe, and culture medium was added ad 7 ml.

6. This cell suspension was usually divided 1:3 - 1:6 onto new plates so that

approximately 1 - 2 million cells per plate were seeded.

3.3.1.2 Freezing and thawing

For production of PC12 stock aliquots, cells were harvested, pelleted and resuspended in 1 ml

freezing medium (about two million cells per ml). Freezing medium consisted of cell culture

medium adjusted to 10% FCS and supplemented with 10% DMSO. The cell solution was


35

aliquoted in 2 ml cryotubes, transferred to ice for 35 min, incubated at -20° C for 45 min and

finally stored at -80° C.

PC12 cells were stored in aliquots at -80°C. When the cells had been passaged 20 times, they

were discarded and a stock aliquot was quickly thawed in a 37° C water bath. The cell

solution was pipetted slowly into 10 ml of FCS and centrifuged briefly at 1000 x g. The pellet

was resuspended in cell culture medium containing twice the amount of serum than usual, and

divided onto two 10 cm plates. Since DMSO is toxic for the cells, the nutrient solution was

replaced by normal cell culture medium after 24 h. PC12 cells from no earlier passage than P6

were used for experiments.

3.3.2 Applied stimuli

For experiments, PC12 cells were seeded on new plates: 20 cm collagen-coated plates for

mitochondrial isolations, 6 well plates for FACS analyses, 24 well plates for ROS

experiments in the Fluoroskan Ascent® plate reader, and 10 cm plates for all other studies.

Incubation time in the new plates was 2 – 3 days, since this time is needed for PC12 cells to

express all relevant channels, transporters etc. (Shafer et al., 1991). When splitting cells for

experiments medium with reduced serum content (5% FCS) was used.

3.3.2.1 6-hydroxydopamine (6-OHDA)

Stock solutions of 10 mM and 50 mM 6-hydroxydopamine were freshly prepared in an

aqueous solution of 0.02% ascorbic acid to prevent immediate autoxidation

(Hayakawa, 1999). Working concentrations of 10 µM, 25 µM, 50 µM, 100 µM and 200 µM

were obtained by adding the appropriate volume of stock solution to the medium at the

desired time prior to harvesting the cells for analysis.

3.3.2.2 1-Methyl-4-phenylpyridinium ion (MPP+)

1-Methyl-4-phenylpyridinium (MPP+) iodide was chosen to compare antioxidant effects of

methysticin, luteolin, resveratrol and tBHQ to the 6-OHDA neurodegenerative PD model.

MPP+ is the metabolite that is generated from 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine

(MPTP) by monoamine oxidases (MAO-B) in the central nervous system (Heikkila et al.,

1984). MPTP has been found to induce Parkinsonian-like symptoms (Langston et al., 1984a;

Langston et al. 1984b) and hence, MPTP and MPP+ have been used as classical agents to

investigate the etiology of the disease in in vitro models (D’Amato et al., 1986). MPP+ stock


36

solution was composed of 50 mM MPP+ in DDW, freshly prepared, and diluted to 50 µM,

100 µM or 200 µM.

Figure 4. Applied stimuli (Source: Wikipedia.org, modified)

3.3.2.3 Staurosporine (STS)

Staurosporine (STS), isolated from the bacterium Streptomyces staurosporeus in 1977,

possesses biological activities ranging from anti-fungal to anti-hypertensive (Rüegg and

Burgess, 1989). The main feature of STS is the inhibition of a variety of protein kinases like

protein kinase C (Tamaoki et al., 1986). It is frequently used for its ability to induce cellular

death via the mitochondrial apoptotic pathway, which includes the release of cytochrome c,

caspase activation, intracellular ROS accumulation, and an increase in [Ca2+]i (Krohn et al.,


37

1998; Prehn et al., 1997; Kruman et al., 1998). STS was used for setting up the flow

cytometrical cell viability assay and as a positive control for increased ROS production. In

those experiments, STS was administered at a concentration of 1 µM, prepared from a 1 mM

stock solution in DMSO (so that the final DMSO concentration would not exceed 0.1%).

3.3.2.4 Valinomycin

Valinomycin is a cyclopeptide neutral ionophore synthesized by Streptomyces fulrissimus.

This ionophore selectivity for K+ renders biological membranes permeable to this cation and

gives the compound the utility as an antibiotic (Cossarizza et al., 1993; Pressman, 1976). In

research Valinomycin is used to uncouple oxidative phosphorylation and disrupt the

mitochondrial membrane potential (∆ΨM) (Cossarizza et al., 1996; Sureda et al., 1997). In

this study Valinomycin was used in a concentration of 1 µM diluted from a 2 mM stock

solution in ethanol as a positive control for ∆ΨM experiments.

3.3.3 Protection of PC12 cells

3.3.3.1 The JNK inhibitor SP600125

SP600125 (SP) is an anthrapyrazolone ATP-competitive inhibitor of JNK. Although it exerts

a great specificity towards JNK, also other ERK and p38 kinases, the closest relative kinases,

and the upstream kinases MKK4 and 7 can be targeted due to the similarity of the ATP

binding site (Bogoyevitch et al., 2004). SP was applied 30 min prior to stimulation with

6-OHDA. When 25 µM or 50 µM 6-OHDA were administered SP concentration was 2 µM,

for this was found to be the minimal effective dose to inhibit the phosphorylation of the

transcription factor c-Jun, the major target of JNK activity (Bennett et al., 2001; Eminel et al.,

2004). Only at higher 6-OHDA concentrations (50 µM to 200 µM) SP600125 application was

adjusted when indicated.

3.3.3.2 Methysticin

Methysticin is one of 6 major kavalactones yielded from Piper methysticum (kava-kava).

Effects of kavalactones include mild sedation, improved cognitive performance and lift of

spirits (Thompson et al., 2004). Muscle relaxant, anaesthetic, anticonvulsive and anxiolytic

effects are thought to result from direct interactions of methysticin with voltage-gated sodium

channels (Magura et al., 1997). Furthermore, methysticin reduces infarct size in mouse brains


38

after ischemic insult by middle cerebral artery (MCA) occlusion in a comparable manner to

memantine (Backhauss and Krieglstein, 1992). There are hints that methysticin could reduce

oxidative stress, as it inhibits monoamine oxidase B (MAO-B), a source for oxygen radicals

in the cytoplasm, and showed moderate radical scavenging abilities (Uebelhack et al., 1998;

Wu et al., 2002). However, long-term kava use is associated with substantial alterations in

liver enzymes and liver toxicity (Mathews et al., 2005). In this study methysticin was used in

a final concentration of 25 µM, taken from a 25 mM stock solution in DMSO.

Figure 5. Protective agents (Source: Wikipedia.org, modified)


39

3.3.3.3 Luteolin

Luteolin, a 3′,4′,5,7-tetrahydroxyflavone, is usually found in a glycosylated form in e.g.

celery, green pepper, and chamomile tea. Luteolin possesses high DNA protective effect in

the presence of H2O2 (Romanova et al., 2001), anti-inflammatory, antioxidant and

phytoestrogen-like activities (Dall'Acqua and Innocenti, 2004). Luteolin is among the most

potent and efficacious flavonoid inhibitors of lipopolysaccharide (LPS)-induced TNF-α and

IL-6 production, as well as nitric oxide expression (Xagorari et al., 2001). Its neuroprotective

effects result from radical scavenging features and interaction with signaling pathways

promoting cell survival (for review see Dajas et al., 2003). Luteolin, in the final concentration

of 5 µM, prepared from a 5 mM stock solution in 100% ethanol, was used for assays in the

PC12 cell model. This contributed to a study of our research group elucidating that

neuroprotection by luteolin is mediated by the increased activity of the neuroprotective

transcription factor Nrf-2 (Wruck et al., 2007).

3.3.3.4 Resveratrol

Resveratrol (3,5,4'-trihydroxystilbene) belongs to the phytoalexins, antibacterial and anti-

fungal chemicals produced by plants as a defense against infection by pathogens. The

compound is primarily found in the skins of grapes, which has led to a vivid discussion about

the contribution of resveratrol to health effects of red wine (Corder et al., 2006). Indeed, there

are many reports about beneficial health effects, such as anti-cancer, antiviral,

neuroprotective, cardioprotective, anti-aging, anti-inflammatory and life-prolonging effects

(for review see Pervaiz, 2003).

On cellular level, mechanisms of resveratrol actions include modulation of transcription

factors, like NF-kB, AP-1 (Jun/Fos) and Nrf-2, alteration of the expression and activity of

cyclooxygenase (COX) enzymes, improvement of mitochondrial functions, antioxidant

activities, inhibition of lipid oxidation, induction of cell death by release of pro-apoptotic

mediators from mitochondria, increase in the expression of p53, inhibition of cyclin-

dependent kinase (cdk), cell cycle arrest, inhibition of the JNK pathway (Pervaiz, 2003; Hsieh

et al., 1999; Manna et al., 2000; Kutuk et al., 2006; Leiro et al., 2005; Plin et al., 2005;

Lagouge et al., 2006; Wruck et al., 2007). Resveratrol was applied in a final concentration of

5 µM, prepared from a 5 mM stock solution in DMSO.


40

3.3.3.5 tert-Butylhydroquinone (tBHQ)

TBHQ is a highly effective antioxidant used to enhance storage life of goods and food. In

research it is a standard compound to compare antioxidant efficacy. tBHQ was applied in a

concentration of 5 µM, prepared from a 25 mM stock solution in DMSO.

3.3.4 Trypan blue viability staining

Various manipulations of cells, including splitting, freezing and stimulations may provoke

cell death. To determine the number of surviving cells in a given population, exclusion of the

dye trypan blue was used. Healthy cells are able to exclude this dye for a certain time, but

trypan blue will quickly diffuse into the cells which have lost their membrane integrity. The

following protocol was used:

Protocol:

1. Cells were washed with PBS (37° C)

2. Cells were rinsed with 2 ml 0.5 M EDTA in PBS (37° C) and placed for 2 min in the

incubator (37° C, 5% CO2)

3. 5 ml of medium were added (37° C)

4. Cells were scraped off the cell culture plate and transferred into a 15 ml tube

5. Cells were centrifuged for 10 min (1000 x g, RT)

6. After centrifugation, the pellet was thoroughly resuspended in an appropriate amount

of PBS

7. 20 µl of the cell suspension were mixed with 20 µl of trypan blue solution and

transferred to a hemocytometer twin chamber. Living cells in the 16 squares of both

chambers were counted, and the percentage of viable cells was determined.

3.3.5 Preparation of mitochondria

Mitochondria from PC12 cells were isolated adapting the method described by Kapirnich

et al. (Kapirnich et al., 2002). All steps were conducted at 4° C.

Protocol:

1. Cells plated on 20 cm cell culture plates were washed twice with PBS and harvested.

2. Centrifugation for 6 min (1000 x g, 4° C).


41

3. Pellets were resuspended in 4 ml of PBS and living cells were counted with trypan

blue.

4. Cells were centrifuged again; pellets were resuspended in sucrose buffer (for 5 x 106

cells 100 µl sucrose buffer) and transferred into 1.5 ml tubes. The cell solution was

stored for 1 h on ice.

5. After 1 h cells were lysed by aspiration through a 27 gauge syringe (25 - 30 times).

6. Lysates were centrifuged for 5 min (750 x g, 4° C).

7. Pellets containing mainly nuclear proteins were washed, lysed in DLB-buffer and

frozen at -80° C. Supernatants were collected into a sterile 1.5 ml tube and centrifuged

for 15 min (10,000 x g, 4° C).

8. Supernatants containing the cytoplasmic proteins were transferred into a sterile 1.5 ml

tube and SDS from a 10% stock solution was added to a final concentration of 1%

SDS.

9. Pellets containing mitochondrial extracts were resuspended in sucrose buffer and

centrifuged for 15 min (10,000 x g, 4° C). This washing step was repeated once more.

10. Mitochondrial pellets were lysed in DLB-buffer (Tables 5).

11. Cytoplasmic and mitochondrial extracts were boiled for 5 min at 95° C.

12. Mitochondrial extracts were sonicated twice for 5 sec and centrifuged (15 min,

13,000 x g, 4° C) to remove insoluble materials.

13. Cytoplasmic and mitochondrial extracts were stored at -80° C.

Table 4. Composition of sucrose buffer

Sucrose buffer

HEPES 20 mM

KCl 10 mM

MgCl2 1.5 mM

EGTA 1 mM

EDTA 1 mM

Sucrose 250 mM

DTT 1 mM

PMSF 0.1 mM

Freshly prepared and kept at 4° C.


42

Samples for the assessment of the changes of the cytosolic protein ß-actin and the

mitochondrially resident enzyme cytochrome c oxidase subunit IV (COX IV) were taken as

follows: whole cell sample during step 3 (controls, C), resuspended mitochondrial suspension

samples during washing step 9 (intermediate fractions, I1 and I2), and the final mitochondrial

fraction (M).

3.3.6 Protein identification by Western blot

3.3.6.1 Denaturing protein extraction

Denatured protein extracts from mitochondria were prepared using a Tris-buffered sodium

dodecyl sulfate (SDS) lysis buffer (denaturing lysis buffer).

1. The cell pellet collected in a 1.5 ml tube was resuspended in 50 - 300 µl of lysis buffer

(depending on the size of the pellet).

2. The cell solution was incubated for 5 min at 95° C in a heating block.

3. The samples were sonicated twice for 5 s to disrupt the cells by cresting vibrations

which cause mechanical shearing of the cell wall.

4. Insoluble material was removed by centrifugation (15 min; 13,000 x g; 4° C).

5. The supernatant was transferred into a new 1.5 ml tube and stored at -80° C.

Table 5. Composition of denaturing lysis buffer.

Denaturing lysis buffer (DLB-buffer)

Tris 10 mM

SDS 1%

Phosphatase inhibitor Cocktail II 1%

3.3.6.2 SDS-PAGE

Polyacrylamide gels were prepared by co-polymerization of acrylamide monomers with the

cross-linker bis-acrylamide. The reaction was catalyzed by ammonium persulphate (APS) and

initiated by N,N,N’,N’-tetramethylethylenediamine (TEMED). For better resolution, a short

stacking gel was set on top of the main resolving gel. Differences in composition between

these two gels resulted in concentration of the protein samples into narrow bands in the


43

stacking gel and separation of the bands according to their size in the resolving gel (Tables 6

and 7). For preparation of the resolving gel different percentages of acrylamide were used,

depending on the molecular weight of protein to be analyzed. 15% resolving gel was used to

detect cytochrome c (cyt c), and cytochrome c oxidase subunit IV (COX IV), all other

proteins were analyzed in a 12% resolving gel (Tables 6 and 7).

Table 6. Composition of solutions used in SDS-PAGE

Resolving buffer Stacking buffer

Tris, pH 8.8 1.5 M Tris, pH 6.8 0.5 M

SDS 0.4% SDS 0.4%

MgCl2 1.5 mM Sucrose 250 mM

PMSF 0.1 mM DTT 1 mM

Stored at 4° C Stored at 4° C

Acrylamide/bis-acrylamide solution Electrophoresis buffer (10x)

Acrylamide 30% Tris, pH 8.3 0.25 M

Bis-acrylamide 0.8% Glycine 1.92 M

SDS 1%

Stored at 4° C in the dark Stored at RT

Sample buffer (5x)

Tris, pH 6.8 312.5 mM

SDS 10%

2-Mercaptoethanol 10%

Glycerol 50%

Stored at 4° C

3.3.6.3 Preparation of polyacrylamide gels

The MiniProtean II Vertical PAGE chamber and glass plated were set up. Resolving gel

monomer solution combining all reagents containing TEMED and APS was prepared. The

solution was carefully introduced to minimize possibility of air bubbles trapped within the


44

gel. When the appropriate resolving gel solution was added, the gel was over-layed with

DDW to keep the gel surface even and allowed to polymerize for 10-30 min at RT. After

polymerization, a distinct interface appeared between the separating gel and DDW which had

to be removed. Subsequently, the stacking gel (Table 7) was prepared and pipetted over the

polymerized resolving gel until the solution reached top of front plate. Immediately, a 10- or

15-well comb was inserted into gel plates until bottom of teeth reach top of front plate. It is

important to be sure that bubbles were not trapped on the ends of teeth. The stacking gel was

allowed to polymerize within 30 min at RT.

After the stacking gel had polymerized the comb was removed carefully and the gel plates

were placed into the electrophoresis chamber. The chamber was filled with 1x electrophoresis

buffer and the wells were cleaned from residual gel particles.

Table 7. Composition of separating gels for SDS-PAGE

Target protein size < 30 kDa 30-60 kDa

Component 15% gel 12% gel

Acrylamide 6.7 ml 4 ml

4 x resolving buffer 2.3 ml 2.5 ml

Autoclaved DDW 2.3 ml 3.4 ml

TEMED 10 µl 10 µl

10% APS 100 µl 100 µl

Composition of the 3% stacking gel for SDS-PAGE

Component Quantity

Acrylamide 0.6 ml

4 x stacking buffer 1.5 ml

Autoclaved DDW 3.9 ml

TEMED 6 µl

10% APS 60 µl


45

3.3.6.3 Preparation of protein samples

Protein samples, used for Western blot were diluted with autoclaved DDW in order to obtain

20 µg of total protein in a volume of 8 µl. 2 µl of 5x SDS sample buffer were added, and the

samples were heated to 95° C for 5 min. Then the samples were loaded onto the gel. To

estimate the molecular weights of bands detected by Western blotting, broad range prestained

protein marker was used in the most left and/or most right lane.

3.3.6.4 Electrophoresis

The samples and the protein marker were carefully loaded into the cleaned wells and run

through the stacking and resolving gels at a constant current of 30 mA. Usually, gels were run

for 30 min after the tracking dye had passed the end of the gel. Finally, gels were removed

from the gel plates, the stacking gel was discarded, and the resolving gel was used for

Western blotting.

3.3.6.5 Immunoblotting

Table 8. Solutions for Western blotting experiments

TBS (10x) TTBS

Tris 200 mM TBS (1x)

NaCl 1.37 mM Tween-20 1 ml/l

Stored at 4° C Stored at 4° C

Anode Buffer I Anode Buffer II

Tris 30 mM Tris 300 mM

Methanol 20% Methanol 20%

Stored at RT Stored at RT

Cathode Buffer

Tris 300 mM

Methanol 20%

6-aminocaproic acid 10%

Stored at RT


46

In Western blotting experiments proteins are transferred from a SDS-PAGE gel to a synthetic

membrane, and blotted proteins are detected by specific antibodies. Prior to addition of

antibodies, the membrane is coated with blocking solution (BS), e.g. 4% non-fat milk or 4%

BSA in TTBS, to avoid non-specifical binding to the membrane. The primary IgG (e.g.

produced in mice) antibody recognizes the protein of interest while the secondary antibody

recognizes the Fc region of the first antibody. This secondary antibody is coupled to an

enzyme, e.g. a horseradish peroxidase (HRP), which converts a chemiluminescence substrate.

After the proteins were separated by SDS-PAGE, they were transferred to a polyvinylidene

difluoride (PVDF) membrane by semi-dry blotting using an electroblotter. Three different

transfer buffers were used (anode buffer I and II, cathode buffer). The PVDF membrane

which was cut to the size of the gel (9 x 6 cm) was activated in 100% methanol for 3 min.

Subsequently the membrane was rinsed in DDW for 2 min and equilibrated in anode buffer I

until use. For each gel, 15 pieces of blotting paper were cut to the size of the gel (9 x 6 cm).

6 pieces of them were pre-soaked in anode buffer II and placed on a glass plate. Three pieces

of blotting paper were pre-soaked in anode buffer I and placed over the anode

buffer II-soaked filter paper. The equilibrated membrane was then placed over the filter paper

and the gel was placed in close contact with membrane after pre-soaked in cathode buffer.

The ‘sandwich’ was completed by stacking remaining 6 pieces of filter paper pre-soaked in

cathode buffer. Finally, the blotting ‘sandwich’ was turned upside down and placed into the

semi-dry transfer unit in which the lid is the cathode.

Proteins were transferred to the PVDF membrane using a constant current of 0.8 mA/cm2 for

1 h (protein size 30-80 kDa) or 1.5 h (>80 kDa). After transferring the protein to the

membrane, the membrane was washed for 20 min in 1x TTBS buffer and blocked with

blocking solution for 60 min. Subsequently the membrane was incubated with the first

antibody at 4° C overnight (see also Table 9). The next day, the primary antibody solution

was discarded and the unbound antibodies were removed by washing the membranes once for

15 min and twice for 5 min each with TTBS. Usually the membrane was incubated with a

dilution of 1:4000 goat-anti-rabbit secondary antibody or with a dilution of 1:3000 goat-anti-

mouse secondary antibody for 30 min at RT. After discarding the secondary antibody

solution, the membrane was washed once for 15 min and three times for 5 min each with

TTBS.


47

Table 9. Primary and secondary antibodies for western blots

Antibody Dilution and Buffer Source Manufacturer

COX IV 1:800 in 4% BS rabbit CST

Cytochrome c 1:5000 in 4% BS mouse BD

Grp75 1:5000 in TTBS mouse Stressgen

JNK1 1:1000 in TTBS mouse Pharmingen

JNK2 1:1000 in 4% BS mouse Santa Cruz

JNK3 1:1000 in TTBS rabbit Alexis

JIP-1 1:1000 in 4 % BS rabbit Santa Cruz

MKK4 1:500 in TTBS rabbit Santa Cruz

MKK7 1:1000 in 2% BS rabbit Santa Cruz

phospho-JNK 1:2500 in 4% BS rabbit Promega

phospho-MKK4 1:500 in TTBS rabbit Santa Cruz

total JNK 1:1000 in TTBS rabbit CST

ß-actin 1:5000 in TTBS mouse Sigma

3.3.6.6 ECL-reaction

After the last washing step, the membrane was placed with the protein side up on a glass

plate. For a 6 x 9 cm (standard sized) membrane, 1 ml of ECL Plus HRP substrate was

prepared immediately prior to use by mixing 0.975 ml of ECL Plus Reagent A with 25 µl of

ECL Plus Reagent B. The membrane was carefully covered with the HRP substrate solution

and incubated for 3 min. Subsequently, all the HRP substrate was allowed to drip off the

membrane and the membrane was placed inside the plastic pocket of a film cassette. The

chemiluminescence on the membranes was detected by exposing the membranes to Hyperfilm

ECL films in a darkroom. Films were developed and fixed by a film processor.

3.3.6.7 Stripping of Western blot membranes

After Western blot and detection of the protein, the membrane can be used to detect an other

protein. For stripping, the membrane was incubated with stripping solution (Tris 62.5 mM,

SDS 2%, 2-Mercaptoethanol 100 mM) for 30 min at 50° C and 25 rpm in an incubator. After

stripping, the membrane was washed twice for 10 min each with TTBS and blocked with

blocking solution. Subsequently, the membrane was incubated with primary antibody.


48

3.3.6.8 Ponceau S staining of Western blot membranes

Ponceau S is the only staining method which is completely compatible with all procedures of

immunological probing, because the stain is transient and can be washed away so that it does

not interfere with subsequent detection of antigens. After the ECL reaction, membranes were

washed twice with TTBS and then stained with Ponceau S for 20 min. The staining solution

was re-used several times. Stained membranes were washed twice with DDW for 5 min each

before air-drying.

3.3.7 Basic principles of flow cytometry

Flow cytometry is a sensitive and rapid method for the detection of single cells. The term

derives from the measurement of single cells that are passed through a thin capillary in

solution. In the flow chamber the suspension is hit by a laser beam, and cells/organelles that

pass the beam lead to scattering of the laser light, which is detected at two different angles:

the forward scatter (FSC) at low angle and the sideward scatter (SSC) at 90°. The intensity of

the FSC signal is related to the size of the cell: large cells lead to a higher scattering than

small cells. On the other hand the intensity of the SSC signal is related to the morphology of

the cell: cells with high granularity exhibit higher sideward scatter than cells with little

cellular structure. The intensities of FSC and SSC thus allow for identification of different

cell populations and also for identification of intact cells, since apoptotic cells exhibit smaller

size and higher granularity. Furthermore, cells can be stained with different fluorescent dyes

that are excited by the laser light and emit fluorescent light which is also detected at 90°. The

optical system contains different filters which allows for simultaneous detection of several

different dyes in a single cell. Figure 6 shows a schematic representation of the optical system

in the Becton Dickinson FACSCalibur system.

The FACSCalibur system contains a 488 nm blue laser, photodetectors for FSC and SSC with

488 nm filters as well as photodetectors for three different fluorescence channels with filters

of 530 nm (green), 585 nm (orange) and 650 nm (red). These different wavelengths are

termed channels FL1, FL2 and FL3 respectively. A single cell can therefore be characterized

by up to five signals from FSC, SSC and the three fluorescence channels. Although the

detectors of the different fluorescence channels are equipped with filters specific for a certain

wavelength range, most fluorescent dyes show a broad emission peak and therefore some

light may be also detected in the adjacent fluorescence channels. This overlap of emission


49

spectra of different dyes has to be corrected by compensation if cells are simultaneously

stained with several dyes. Compensation is the electronic subtraction of unwanted signal to

remove the effects of spectral spillover.

Figure 6. The Beckton Dickinson FACSCalibur system

The cell/organelle suspension is sucked into the flow chamber where fluorescent molecules in the solution are excited by a laser beam. Scattered light is detected in 180° (FSC) and 90° (SSC). Emitted fluorescence is split and narrowed to specific wavelength range and detected at 90° with photomultiplier tubes (FL diodes). (© Beckton Dickinson Inc., modified) Abbreviations: FL1, FL2, FL3 (fluorescence channels 1, 2, 3), FSC (forward scatter), LP (long pass), SSC (side scatter).

3.3.7.1 Flow cytometrical data analysis

For quantification of ROS PC12 cells were stained with the ROS-sensitive dyes 2',7'-dichloro-

dihydrofluorescein-diacetate (H2DCF-DA) and dihydrorhodamine 123 (DHR). For the

characterization of the mitochondrial membrane potential (∆ΨM) samples were incubated with

the dual-fluorescent probe JC-1, and for the detection of apoptotic and necrotic cells Annexin

V-FITC and propidium iodide (PI) were used. All samples were analyzed by flow cytometry

using Becton Dickinson FACSCalibur flow cytometer and Cell Quest Pro software. A

minimum of 10,000 events were recorded per single measurement. Results represent a

number of at least three independent experiments.

PC12 cells were gated according to size and morphology in the SSC vs. FSC density plot

(Figure 7A), and only the gated events were evaluated for the intensities of fluorescent dyes.


50

Due to the small size of mitochondria, only a threshold was set to 60 in the FCS to rule out

any events being counted below that.

Figure 7. PC12 cells (A) and mitochondria (B) in the scatter plot.

3.3.7.2 Staining of PC12 cells with ROS-sensitive fluorescent dyes

In general, 1 x 106/ml PC12 cells were incubated with ROS-sensitive dyes at 37° C in cell

culture medium without phenol red, washed twice and resuspended for FACS analysis in

FACS analysis medium (PBS containing 4.5 g/l glucose and 0.2% EDTA). Cells were

analyzed immediately after staining and always kept on ice in the dark until measurement.

3.3.7.2.1 2′,7′-Dichlorofluorescein (DCF)

2',7'-Dichloro-dihydrofluorescein-diacetate (H2DCF-DA) is a widely used ROS-sensitive dye.

The diacetate enables 2',7'-dichloro-dihydrofluorescein , also termed 2',7'-dichlorofluorescin

(H2DCF), to diffuse across the cell membrane where it is hydrolyzed by intracellular esterases

to the non-fluorescent H2DCF. Consequently, H2DCF-DA was administered to PC12 cells,

and H2DCF was given to samples of isolated mitochondria. In the cell H2DCF is oxidized by

various ROS to the highly fluorescent, 2-electron oxidation product, DCF (Bass et al., 1983;

Rothe and Valet, 1990; Hempel et al., 1999). Mainly hydrogen peroxide in combination with

enzymatic peroxidase activity seems to be responsible for H2DCF oxidation (Walrand et al.,

2003). H2DCF is a useful tool for monitoring cytosolic ROS levels, as well as ROS generated

in the mitochondrial intermembrane space, and ROS that are released from mitochondria into

the surrounding cytosol (Diaz et al., 2003). The emission maximum of DCF is at 525 nm and

can be detected in the FL1 channel.


51

After 20 min of H2DCF-DA preloading, cells were washed twice with PBS and pellets were

resuspended in FACS analysis medium. Analyzing PC12 cells, fluorescence was determined

immediately for controls and stimulated samples. Results were reported as fluorescence

intensity in percent of controls.

3.3.7.2.2 Dihydrorhodamine (DHR)

Dihydrorhodamine (DHR) is the non-fluorescent reduced form of rhodamine. Due to its

lipophilicity it can easily diffuse across cell membranes. Inside the cell, it is oxidized by

various ROS to the positively charged fluorescent rhodamine which is incorporated into

mitochondria dependent on mitochondrial membrane potential (Johnson et al., 1980).

Although several ROS could be responsible for oxidation of DHR (Hempel et al., 1999), it

has been described to be most sensitive towards oxidation by hydrogen peroxide (Walrand et

al., 2003), especially in the presence of cytochrome c oxidase in mitochondria (Royall and

Ischiropoulos, 1993). Thus it can be used as a marker for mitochondrial ROS production. The

emission maximum of rhodamine is at 529 nm which is detected in channel FL1.

Table 10. Overview of the ROS-sensitive dyes, their properties and application

Dye Ex (nm)

Em (nm)

FACS detection channel

Concentration Incubation

time at 37° C

Specificity

H2DCF (DA) 504 525 FL1 10 µM 20 min

peroxides, cytosolic/whole-cell ROS, peroxynitrite

DHR 507 529 FL1 10 µM 30 min

peroxides, hydroxyl radical, mitochondrial ROS, peroxynitrite

3.3.7.3 Assessment of mitochondrial membrane potential with JC-1 The integrity of the inner mitochondrial membrane is determined by a specific potential

gradient (∆ΨM) over this membrane. This can be correlated to the uptake of the cationic

carbocyanine dye JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazol-carbocyanine

iodide) into the matrix (Reers et al., 1991; Smiley et al., 1991; Cossariza et al., 1993). The

mitochondrial membrane potential, across the inner membrane, determines the redistribution

of this dye, which depends on the transmembrane electric field (negative inside of about

180 - 200 mV) and the concentration gradient of the dye.


52

The fluorophore JC-1 has the property that when excited at 485 nm (or in flow cytometry

488 nm by an Argon laser), the emission spectrum will be dependent on the concentration of

the molecule. In dilute solutions of <300 nM it gives a green fluorescence at 527 nm, but

raising the concentration over 1 mM leads to the appearance of a very strong red-orange

fluorescence (at 590 nm, Invitrogen data sheet). This is due to the formation of aggregates of

the dye, named J-aggregates. In a mitochondrial matrix, bounded by an inner membrane with

a large ∆ΨM, the dilute external concentration of the dye is concentrated to a level that enables

the formation of J-aggregates.

JC-1 can be used as an indicator of mitochondrial potential in a variety of cell types, including

PC12 cells (Dispersyn et al., 1999; Nuydens et al., 1999), as well as in isolated mitochondria

(Cossariza et al., 1996). JC-1 is more specific for mitochondrial versus plasma membrane

potential, and more consistent in its response to depolarization, than other cationic dyes such

as [DiOC6 (3)] and rhodamine (Salvioli et al., 1997). The ratio between red versus green

fluorescence is often used as a measure to describe the ∆ΨM and integrity. In fact, measuring

the red fluorescence alone is a superior way to represent ∆ΨM, since the J-aggregates are

specific for mitochondria whereas the monomers’ distribution in the rest of the cell is

heterogenous to its hydrophobic interaction (for review see Bernardi et al., 1999).

PC12 cells and isolated mitochondria were subjected to the following protocol for

determination of the ∆ΨM by the Becton Dickinson FACSCalibur flow cytometer (Table 11).

This procedure is a fixed-point assay measuring the uptake of JC-1 with formation of the

J-aggregates. It is also possible to follow the uptake with time using a kinetic program. The

observed fluorescence of the solution rises to a plateau after 5 - 10 min.


53

Table 11. Protocol for the assessment of the mitochondrial membrane potential (∆ΨM)

PC12 cells Mitochondria

1. PC12 cells were incubated in culture

medium without phenol red and

stimulated with 50 µM 6-OHDA or

1 µM valinomycin (positive control)

for various time points

Mice brain mitochondria were

isolated as described in section

3.3.13.

2. Cells were washed on the plate with

PBS, and collected in PBS at a

concentration of 106 cells/ml

A 50 µl aliquot of the mitochondrial

suspension was diluted in 1 ml PBS.

3. 10 µM JC-1 was added, a concentration that had proven to be optimal regarding

both fluorescence emission wavelengths (strong red and low green FI).

4. Samples were incubated 20 min in the dark at RT.

5. Samples were centrifuged briefly and pellets were resuspended in 1 ml PBS.

6. PC12 cells were centrifuged briefly

and pellets were resuspended in 1 ml

FACS analysis medium

Mitochondria were centrifuged

briefly and pellets were resuspended

in MSH buffer. 10 µM 6-OHDA or

1 µM Valinomycin was added for

the desired duration.

Mitochondria were washed and

resuspended in 1 ml FACS analysis

medium

7. The flow cytometer was calibrated with a non-stained and a stained control.

8. Fluorescence in FL1 and FL2 channels was measured for all samples applying

voltages of 320 - 380 V for FL1 and FL2 each.

9. For PC12 cells the FSC was set to

E-1 (1.00), SSC to 350 V (1.00).

Compensations were applied as

follows:

FL1-FL2 12%, FL2-FL1 28%

For isolated mitochondria the FSC

was set to E-1 (7.00), SSC to 380 V

(1.00). Compensations were applied

as follows:

FL1-FL2 20%, FL2-FL1 35%


54

3.3.7.4 Detection of apoptosis and necrosis in the flow cytometer

Apoptosis can be easily detected by staining with Annexin V and flow cytometrical analysis.

Annexin V is a Ca2+-dependent protein that binds to phospholipids, with a high affinity

towards phosphatidylserine, normally located on the inside of the cell membrane of a living

cell. During apoptosis phosphatidylserine is translocated to and presented on the outer cell

surface. In this very early apoptotic stage, Annexin V coupled with FITC fluorophore

(Ex.: 488 nm, Em.: 518 nm) can detect these cells. The cell membrane of necrotic cells

becomes permeable for large molecules, making it possible for Annexin V to enter necrotic

cells and stain them as well. To distinguish apoptotic from necrotic cells, propidium iodide

(PI) fluorescence is measured from the same sample, a dye that only is detected in non-viable

cells in flow cytometry. PI intercalates into the major groove of the DNA producing a highly

fluorescent adduct. The dye can be excited by the FACS Argon laser (488 nm) and its

fluorescence can be detected from 550 nm up to 670 nm. Despite the broad emission spectrum

it still can be used in combination with other 488 nm-excited fluorophores like FITC, but this

requires a proper compensation of the channels (Pigault et al., 1994; Koopman et al., 1994;

Vermes et al., 1995; van Engeland et al., 1996).

In flow cytometrical analysis the dot plot of Annexin V-FITC and PI fluorescent events can

be divided into four quadrants in the way that healthy cells, apoptotic cells and necrotic cells

would be found in one specific quadrant. Living cells are Annexin V negative and PI negative

(lower left quadrant, LL), apoptotic cells are Annexin V positive but PI negative (lower right

quadrant, LR), and necrotic cells are both positive (upper right quadrant, UR) (Figure 8). The

position where to set the quadrant was determined beforehands with healthy cells, saved and

re-used in a set of three independent experiments. Events with strong PI but low Annexin V

fluorescence could be cells that are necrotic and permeable for PI, but not yet for Annexin V.

The Stats option in the Cell Quest program provided the exact percentages of each quadrant.

The number of events in the lower left quadrant of the dot plot (healthy cells) was taken for

each sample, and presented as bar charts, i.e. the change in viable cell number.


55

Figure 8. Quadrant setting for PC12 cell apoptosis and necrosis

In the preparation of Annexin V-FITC/PI fluorescence detection, PC12 cells were incubated

with 50 µM 6-OHDA for 4, 8, 12, 16, and 24 h; and, in a second experiment, with 10, 25, 50,

100, and 200 µM 6-OHDA for 24 h. After that, cells were washed twice with cold PBS,

centrifuged 5 min at 500 x g (4° C) and resuspended in 1x binding buffer (1 part 10x binding

buffer diluted in 9 parts DDW) at a concentration of 1 x 106 cells/ml. According to the

manufacturer’s protocol, 5 µl Annexin V-FITC (purchased solution, 50 µg/ml in 50 mM Tris-

HCl, pH 7.5, containing 100 mM NaCl, Sigma) and 5 µl PI (final concentration 2 µg/ml) were

added to 490 µl of the cell suspension. Samples were incubated 15 min in the dark and gently

vortexed from time to time and then analyzed with the flow cytometer.

Table 12. Settings for the detection of apoptosis and necrosis

FSC E01 1.00 Lin

SSC 350 1.00 Lin

FL1 480 1.00 Log

FL2 500 1.00 Log

Threshold: FSC 20

Compensation: FL2-FL1 (35%)


56

Table 13. Buffers for the detection of apoptosis and necrosis

PI stock solution 10x Binding buffer

PI 1 mg/ml HEPES, pH 7.5 100 mM

KH2PO4 10 mM NaCl 1.4 M

NaCl 150 mM CaCl2 25 mM

Stored at 4° C, protected from light Sterile filtered. Stored at 4° C

3.3.8 Experiments using the fluorescence microplate reader

The Fluoroskan Ascent® microplate reader was used to determine ROS generation in PC12

cells. In principle, emitting light from a quartz halogen lamp passes an excitation filter that

selects only light from a certain wavelength. The sample in the multi-well plate is irradiated

with this light, and the fluorescent dyes in the samples produce an emission spectrum at

longer wavelengths which is detected in a 90° angle to reduce background signals from

scattered light, also passing through a filter selected for the expected emission maximum (see

Figure 9).

Figure 9. General measuring principle of fluorescence plate readers. (©BioTek

Instruments Inc., Winooski, USA)


57

In this study isolated mitochondria and PC12 cells were tested for their ROS levels with the

aid of DCF. Six aliquots of a suspension of isolated mitochondria were dispensed in six tubes

(120 µl in 1 ml MSH buffer per tube). Samples were preloaded with 10 µM DCF for 60 min

(4° C), and washed/centrifuged after this time. 6-OHDA was given 60 min (4° C) in various

concentrations (10 µM, 25 µM, 50 µM, 100 µM and 200 µM) and washed/centrifuged after

this time. Each pellet was resuspended in 4 ml TBS and divided into 4 wells of a 24 well plate

(24 well Nunc multidish). DHR fluorescence of controls and stimulated samples was

measured immediately. Results were normalized to control protein levels and displayed as

mean fluorescence intensity of four wells (in percent of control). A total number of three

independent experiments were conducted.

To examine the effect of JNK inhibition on ROS generation following 6-OHDA, PC12 cells

were preloaded with 10 µM H2DCF-DA for 30 min, and hereafter, washed once with TBS.

Then 6-OHDA with or without the JNK inhibitor SP600125 was applied for 60 min.

SP600125 was added according to the following scheme (Table 14).

Table 14. Treatment regimen to test the effect of JNK inhibition on ROS levels

6-OHDA - 10 µM 25 µM 50 µM 100 µM 200 µM

SP600125 2 µM 2 µM 2 µM 2.5 µM 3 µM 4 µM

Cells were washed once with prewarmed TBS, the pellets obtained after centrifugation were

resuspended in 4 ml TBS for each sample and divided into four wells of a 24 well plate. DCF

fluorescence was measured using the following filters (Ex: 485 nm, Em: 538 nm). A cell

suspension aliquot from each sample was taken for cell count, fluorescence intensities were

standardized to the number of control cells and results were normalized to control protein

levels and displayed as percent of control.

Furthermore, the antioxidant effects of methysticin, luteolin, resveratrol and

tert-butylhydroquinone (tBHQ) on 6-OHDA or MPP+-induced ROS elevation were

investigated. PC12 cells were incubated with these antioxidants for 16 h, after that they were

washed and preloaded with H2DCF-DA (10 µM) for 30 min, and stimulated with 50 µM

6-OHDA or 100 µM MPP+ for 60 min. Cells were washed once with prewarmed TBS, the

pellets obtained after centrifugation were resuspended in 4 ml TBS for each sample and


58

divided into four wells of a 24 well plate. DCF fluorescence was measured using the

following filters (Ex: 485 nm, Em: 538 nm). A cell suspension aliquot from each sample was

taken for cell count, fluorescence intensities were standardized to the number of control cells

and results were displayed as percent of control.

3.3.9 Experiments using the spectrofluorometer

In brief, the basic principle of fluorometry is that light from an excitation source passes

through a filter or monochromator, and strikes the sample. A proportion of the incident light

is absorbed by the sample. The fluorescent light is emitted in all directions. Some of this

fluorescent light passes through a second filter or monochromator and reaches a detector,

placed at 90° to the incident light beam to minimize the risk of transmitted or reflected

incident light reaching the detector. Classical fluorometry was performed due to the

advantage that fluoresence intensities of a sample could be monitored on-line. The instrument

used was a monochromator, which allowed for the selection of single excitation and emission

wavelengths. For experiments with the dual-fluorescent JC-1, the excitation wavelength was

chosen to be 488 nm, the emission wavelengths were 530 nm and 590 nm.

100 µl PC12 cell suspension were added to 400 µl PBS, a suspension of isolated mitochondria

was diluted 1:20 in 500 µl PBS. H2DCF-DA (for cells) and H2DCF (for mitochondria) was

given at a concentration of 10 µM and incubated for 20 min. Samples were washed twice,

transferred to a quartz cuvette and inserted into the appropiate slot in the fluorometer. When a

linear baseline was gained (800 - 1400 sec), the excitation of the probe was terminated by

closing the shutter, and 6-OHDA was pipetted carefully into the cuvette (50 µM for cells,

10 µM for isolated mitochondria; a total volume of 20 µl was added each). After the injection

of the toxin the measurement was additionally paused for 30 sec to minimize fluorescence

signal disturbances caused by turbulences in the solution. Then the shutter was opened and

measurement continued until the new slope was linear. 1 µM valinomycin served as a control

substance to disrupt the ∆ΨM. JC-1 ratio of the emission wavelengths (590 nm/530 nm) was

calculated and saved simultaneously, and was visualized after the experiment was completed.


59

3.3.10 Fluorescence microscopy

3.3.10.1 Principle of confocal laser scanning microscopy

An objective focuses an expanded light-beam originating from a laser source to a small spot

on the sample, at the focal plane of the objective lens. Reflected light from the illuminated

volume of the specimen is collected by the objective and reflected by a beamsplitter towards a

pinhole arranged in front of the detector. In this case the pinhole is responsible for the

confocal characteristics of the system. Information which does not originate from the focus

level of the microscope objective, is faded out by this arrangement. In contrast, light from the

focal plane is directed through a pinhole and registered by the detector (Figure 10). The

advantage of out-fading information from above or below the focal plane enables the confocal

microscope to perform depth-dependent measurements: optical tomography becomes

possible. A genuine 3D-image can be processed by confocal scanning of sequential levels.

Figure 10. Principle of confocal laser scanning microscopy


60

3.3.10.2 Experimental setup

To investigate the subcellular distribution of mitochondria and JNK isoforms in PC12 cells by

confocal microscopy, cells were grown on 2-well glass coverslips (Nunc) and stimulated with

50 µM 6-OHDA for 4 h, the time point, when JNK had previously been identified to

colocalize with mitochondria after 6-OHDA. Samples were subjected to the following

protocol. As a first step, PC12 cells were incubated with MitoTracker® Red CM-H2XRos

(Molecular Probes; ex. 579 nm, em. 599 nm) at the concentration of 200 nM for 30 min.

Protocol:

1. Cells were washed with ice-cold PBS.

2. Cells were incubated with methanol at -20°C for 10 min.

3. Cells were washed with ice-cold PBS.

4. Cells were fixed with 2% para-formaldehyde in PBS, 30 min at RT.

5. Fixed cells were carefully washed twice with TBS.

6. Samples were blocked with a blocking solution of TBS/BSA/Glycine for 1 h at RT.

7. Primary antibodies of JNK1 or JNK2 (or cytochrome c in conventional fluorescence

microscopy) were applied in a dilution of 1:100 in blocking solution.

8. Samples were incubated with primary antibody solution at 4° C over night, protected

from light.

9. Fixed cells were washed with TTBS, 2 x 5 min.

10. Samples were washed with TBS, 3 x 10 min.

11. Secondary antibody (in all cases donkey anti-mouse FITC from Jackson

ImmunoResearch Lab., West Grove, PA, USA) was applied in a dilution of 1:400 in

TBS, and cells were incubated at 37° C for 1 h.

12. Samples were washed with TTBS, 2 x 5 min., and hence 10 min with TBS.

13. Hoechst 33258 dye was added 4 µl per ml, and cells were incubated for 30 min at RT.

14. Samples were washed with TBS, 2 x 5 min.

15. Probes were mounted with SlowFade and sealed.

Confocal laser scanning analysis was carried out with a Zeiss LSM 510 laser scanning

microscope (Carl Zeiss Jena, Jena, Germany). Triple staining pictures represent optical slices

of 0.5 µm. Original magnification was 400x.


61

PC12 cells were stained for cytochrome c using the same protocol, but the protein was

detected by conventional fluoresecence microscopy. The Leica DM L microscope was

equipped with the following filter and reflector systems (cubes):

Table 15. Filter and reflector system of the Leica DM L microscope

Excitation filter (nm) Suppression filter (nm) Detected dye

BP 340 - 380 LP 425 Hoechst 33258

BP 450 – 490 LP 515 FITC

BP 515 – 560 LP 590 Mitotracker Red CM-H2XRos

3.3.11 Breeding of mice

Genetic inactivation of JNK1, JNK2 and/or JNK3 in mice has been described in detail

elsewhere (Yang et al., 1997; Dong et al., 1998; Kuan et al., 1999). JNK knockout mice were

backcrossed once with C57/BL6 wildtype mice. Thereafter, the heterozygous F1 offspring

was crossed to obtain F2 homozygous JNK knockout mice and homozygous wildtype (WT)

controls. This F2 generation was used as parent founders to breed the knockout and WT mice

for animals experiments (F3). This breeding approach minimizes the genetic strain variability

of knockout and control animals on the one hand, and on the other provides the large number

of animals necessary for the different experiments according to international guidelines

(Banburry Conference). All animal experiments have been performed according to the

German and the Finnish law for protection of animals and the NIH guidelines for use and care

of laboratory animals (approval by the Ministerium für Landwirtschaft und Naturschutz, Kiel,

Germany).

3.3.12 Genetic characterization of JNK knock-out mice strains

JNK knock-out mice strains from the animal facility were checked regularly by PCR. Tail

tissue pieces were immediately frozen in liquid nitrogen. In the laboratory surroundings, the

tissue was thawed and trenched with scissors in a semisterile manner. The QIAamp DNA

mini kit was used to conduct the following steps to isolate DNA:


62

Protocol:

1. To each sample 180 µl ATL solution and 20 µl Proteinase K were added, samples

were vortexed, sealed and incubated at 55°C O/N; shaking at 1400 rpm.

2. Epp cups were centrifuged (13,000 rpm; 5 min).

3. Supernatant was mixed with 410 µl AL buffer, transferred to a filter/collection tube

and centrifuged at 6000 x g for 1 min.

4. Filtrate was discarded. Filter was combined with a new collection tube and DNA in

the filter was washed with 500 µl AW1 buffer by centrifugation (6000 x g for 1 min).

5. Filtrate was discarded. Filter was combined with a new collection tube and 500 µl

AW2 buffer was added. Tubes were centrifuged at 6000 x g for 3 min.

6. Filtrate was discarded. Filter unit was plugged in a sterile microcentrifuge tube.

7. 200 µl AE buffer (70° C) was given to the samples. They were incubated for 3 min.

8. Samples were centrifuged (6000 x g; 1 min).

9. Step 7 and 8 were repeated without touching the filtrate.

10. 400 µl filtrate containing DNA was obtained by centrifugation. Filter was discarded.

The concentration of DNA (in ng) was measured using 5 µl sample solution in 1 ml ultrapure

water by assessing the ratio of absorption in the photometer between 280 and 260 nm.

3.3.12.1 Polymerase chain reaction (PCR)

The PCR (polymerized chain reaction) comprise three steps. First, the target genetic material

must be denatured, i.e. the strands of its helix must be unwound and separated by heating to

90-96° C. The second step is hybridization or annealing, in which the primers bind to their

complementary bases on the single-stranded DNA. The preferred annealing temperature for a

PCR depends directly on length and composition of the primers. The third step is DNA

synthesis by a polymerase. Starting from the primer, the polymerase reads the template strand

and matches it with complementary nucleotides. This results in two new helices in place of

the first one, each composed of one of the original strands plus its newly assembled

complementary strand. Frequent repeating of these cycles generates millions of DNA strand

copies.

For PCR 100 ng/µl of DNA was used. PCR Master Mix was prepared freshly according to the

protocol shown in Table 17. Values are for one sample, so upscaling was applied accordingly.

JNK primer mixes were produced by combining the following primers obtained from MWG


63

Biotech/Pharmacia for detection of the complete double-band reaction (Table 16). After

addition of PCR master mix, samples were placed in a thermocycler and PCR program was

started.

PCR-Program

1. 94° C, 2 min

2. 94° C, 30 min

3. 56° C, 30 min (annealing temperature) 29x

4. 72° C, 1 min

5. 4° C, ∞

Table 16. Composition of the JNK primer mixes

Primer Mixes

JNK 1

MJ1B8 (100 µM) 20 µl

MJ1F8 (100 µM) 20 µl

PGKT1 (100 µM) 20 µl

Ultrapure water 73 µl

JNK 2

MJ2B3 (100 µM) 20 µl

MJ2F5 (100 µM) 20 µl

PGKT1 (100 µM) 20 µl


JNK 3

MJ3B3 (100 µM) 20 µl

MJ3F3 (100 µM) 20 µl

PGKP1 (100 µM) 20 µl



64

Table 17. Composition of the PCR Master Mix

PCR Master Mix

10x PCR buffer 5 µl

25 mM MgCl2 5 µl

10 mM dNTP 1 µl

Ultrapure water 34.5 µl

DNA sample 1 µl

Primer Mix JNK 1, 2, 3 3 µl

Taq DNA Polymerase 0.5 µl

3.3.12.2 Detection and analysis of the PCR reaction product

Table 18. Stock solutions required for agarose gel electrophoresis

10x TBE-Buffer 10x Loading Buffer

Tris 9 M Bromphenol blue 0.25%

Boric acid 9 M Glycerol 30%

EDTA 0.2 M TBE 1x

Stored at RT Stored at 4° C

The PCR product is a fragment of defined DNA lengths. A sample from these fragments is

loaded with appropriate molecular-weight markers onto an agarose gel. 1% or 1.5% agarose

gels were prepared with 1x Tris-buffer boric acid-EDTA. The 1x TBE (Tris-boric acid-

EDTA) buffer was set up by diluting 10x TBE buffer with DDW. The required amount of

agarose was dissolved in 1x TBE buffer by heating in a microwave oven. 5 µl/100 ml of a

10 mg/ml ethidium bromide stock solution was added to warm agarose solution and mixed

well. For agarose gel electrophoresis a horizontal gel system was used. The agarose solution

with ethidium bromide was pipetted to this gel system and allowed to polymerize for

20-30 min. When the gel had polymerized, it was transferred to an electrophoresis chamber

filled with 1x TBE buffer. 20 µl DNA samples were mixed with 5 µl 10x loading buffer in

1.5 ml tubes. At the same time 1 µl DNA-ladder was mixed with 5 µl 10x loading buffer and

with 19 µl TBE buffer, resulting in a total volume of 25 µl. DNA-ladder and samples were


65

loaded into wells and the gel was run at 80 mV for approximately 60 min. Bands were

visualized with 312 nm UV light.

3.3.13 Isolation of mitochondria from mice brain

Mice brain mitochondria were isolated according to the following optimized protocol

(sources: Gogvadze et al., 2003; Schild et al., 1996; Zablocka et al., 2003). All steps were

performed on ice, with ice-cold solutions and pre-cooled tubes. Before the experiments, 3%

and 6% Ficoll solutions were prepared from a 20% (w/v) Ficoll (Sigma) stock solution in

Ficoll dilution buffer (see Table 19). For the isolation an overall time of 120 min was

considered. Mitochondrial suspension could be used without any impairment for another

2-3 h when kept on ice.

Table 19. Composition of buffers for mitochondrial isolation

Isolation buffer (IB) Ficoll dilution buffer

Sucrose 320 mM Mannitol 250 mM

EGTA 1 mM Sucrose 60 mM

Tris-HCl pH 7.4 10 mM EGTA 100 µM

Tris-HCl pH 7.5 10 mM

Respiration buffer (RB) MSH buffer

KCl 150 mM Mannitol 210 mM

KH2PO4 1 mM Sucrose 70 mM

Tris 5 mM HEPES 5 mM

Adjusted to pH 7.4 with HCl Adjusted to pH 7.4 with KOH


66

Protocol:

1. Five complete mice brains per group were collected in ice-cold PBS and dissected

under the microscope removing blood vessels and fat. Tissue pieces weighed approx.

1 g.

2. Tissue pieces were washed 3x with ice-cold PBS and then homogenized in 10 ml

ice-cold isolation buffer (IB) (10x tissue weight) in steps with a 2 ml all glass dounce

tissue grinder.

3. Homogenized suspension was collected in a 15 ml Falcon tube and centrifuged at

2000 x g, 3 min (4° C).

4. The upper part of the solution with fatty acids floating and sticking to the tube walls

was carefully taken away.

5. The remaining suspension was resuspended and transferred into a new tube and steps

3 and 4 were repeated once.

6. Supernatant was further processed in 2 ml tubes.

7. Samples were centrifuged at 13,000 x g, 10 min (4° C).

8. Supernatant (cytosol) was collected and frozen at -20° C. The pellets were

resuspended each in 200 µl 3% Ficoll solution and layered onto 1 ml of ice-cold 6%

Ficoll solution.

9. Tubes were centrifuged 20 min at 11,500 x g, 4° C.

10. The final pellet was resuspended in 100 - 200 µl MSH buffer depending on the size of

the pellet, which yielded a protein concentration of 90-100 mg/ml (mitochondria). The

true concentration was measured later by Bradford’s colorimetric assay if necessary.

3.3.13.1 Determination of mitochondrial proteins

A defined volume (30 µl) of isolated mitochondrial suspension was taken and mixed with

300 µl denaturing lysis buffer (DLB, see Table 5). Samples were boiled for 5 min and

immediately used for protein measurement or frozen for later use in Western blot analysis for

assessment of the purity of the mitochondrial fraction. The exact mitochondrial concentrations

were only necessary for fluorometric measurement with the cuvette/plate reader, for

experiments in the flow cytometer, no protein measurement was needed since the instrument

counts the same number of events in all samples.


67

Protein concentrations were determined using Dye Reagent, a variant of Bradford’s

colorimetric assay. To prepare working solution, the Dye Reagent stock solution was diluted

1:5 with DDW. To determine protein concentrations, bovine serum albumin (BSA) was used

as protein standard. Serial dilutions of a 1.2 mg/ml BSA stock solution in autoclaved DDW

(0.1 to 0.6 mg/ml) were performed. Samples were diluted 1:30 or 1:50 in autoclaved DDW.

20 µl of sample, standard and DDW (as blank) were pipetted into disposable plastic cuvettes.

1 ml of the working solution described above was added to each cuvette. All cuvettes were

vortexed to start the reaction and incubated at RT for 10 min. The absorbance at 595 nm

(A595) was measured using a spectrophotometer. The protein concentrations were calculated

based on a standard curve created from in-process standard concentrations of BSA.

3.3.13.2 Estimation of the quality of isolated mitochondria measuring the respiratory

control ratio (RCR)

One of the keystones of the chemiosmotic theory of energy transduction is the impermeability

of the inner mitochondrial membrane to protons. Oxidation of substrates results in extrusion

of protons from the mitochondrial matrix to generate the mitochondrial membrane potential.

High membrane potential suppresses further extrusion of protons and therefore inhibits

respiration. Under resting conditions, the rate of respiration of mitochondria is quite low and

determined by a passive leakage of protons into the mitochondrial intermembrane space.

Phosphorylation of ADP requires translocation of protons into the matrix by a mitochondrial

ATP synthase. This results in a decrease of the membrane potential that stimulates respiration.

Comparison of the respiration rates in the resting state and during phosphorylation of ADP is

a useful measure of the efficiency of mitochondrial functioning. The most reliable and widely

used criterion of the quality of a mitochondrial preparation is the respiratory control ratio

(RCR). RCR is defined as the rate of respiration in the presence of ADP (phosphorylating

respiration, state 3) divided by the rate obtained following the expenditure of ADP (state 4).

An oxygen monitor equipped with a Clark-type oxygen electrode (Oxygraph) was used and

oxygen consumption by intact mitochondria was followed on screen with the supplied

software. Mitochondrial samples were subjected to the following protocol:


68

Protocol:

1. The polarographic system was set up and calibrated to 100% oxygen saturation,

delivering a 1 volt signal. Subsequent steps were performed at 25° C.

2. For testing the mitochondrial quality 2 ml of incubation buffer were placed into the

sample chamber of the oxygen monitor and conditions were set to constant stirring.

An aliquot of mitochondrial suspension (50 µl) was added to a final concentration of

~ 2 mg/ml.

3. After 30 sec 20 µl of 10 mM rotenone was added.

4. The stirring was stopped and the electrode was inserted into the sample chamber. All

air was expelled through the slot in the plunger (slight twisting of the electrode helps

to gather the bubbles at the slot) and the chart speed was set up (10 to 15 mm/min).

5. After 1 min stabilization observed by recorder trace 20 µl of 0.5 M sodium succinate

was added, through the slot on electrode's body with a Hamilton syringe.

6. Mitochondria then started to consume oxygen. The slope of the curve showed the rate

of respiration.

7. After stabilization of respiration (~ 2 min) 10 µl of 50 mM ADP was added. The rate

of respiration than increased (state 3) with subsequent restoration of the initial rate of

respiration (state 4) when all added ADP was phosphorylated.

8. 2 µl of 10 mM 2’,4’-dinitrophenol (DNP) was added. DNP disrupts the mitochondrial

potential that is the basis for oxygen consumption.

9. The rate of respiration was calculated from the recorder trace as the amount of oxygen

consumed in 1 min assuming that at 25° C and normal atmospheric pressure the

concentration of oxygen in the incubation buffer is 250 µM.

10. The RCR was calculated by dividing the state 3 respiration rate by the state 4

respiration rate.

Mitochondria with a respiratory control ratio above 3 were acceptable for use in experiments

and were stable for the next four hours.


69

3.3.13.3 Purity of mitochondrial preparations

The purity of isolated mitochondria from mice brains was assessed by Western blot analysis

of the obtained mitochondrial suspension. After lysis with DLB-buffer (see Table 5),

electrophoresis and blotting, antibodies against the mitochondrial constitutional protein

cytochrome c oxidase subunit IV (COX IV) and ß-actin for the assessment of cytosolic

contaminations were applied to the membranes.

3.3.14 Primary cells

Table 20. Solutions for primary cell culture

Hanks Solution Dissection Solution

Pulver media w/o Mg2+ and Ca2+ Hanks solution

NaHCO3 35 mg/ml BSA 3 mg/ml

HEPES 10 mM MgSO4 1.4 mg/ml

D-Glucose 6 mg/ml

Gentamycin 5 µg/ml

Digestion Solution Culture Media

NaCl 8 mg/ml MEM

KCl 0.37 mg/ml D-glucose 5mg/ml

Na2HPO4 0.99 mg/ml Transferrin 0.1 mg/ml

HEPES 5,95 mg/ml Insulin 25 µg/ml

NaHCO3 0.35 mg/ml Glutamax 2 mM

Gentamycin 5 µg/ml

1st day Culture Media 2nd day Culture Media

Culture media Culture media

FCS 10% FCS 5%

B 27 supplement 2%

AraC 5 µM


70

3.3.14.1 Coating of the plates

For providing attachment and growth of murine primary cells, 4-well plates and culture dishes

(35 mm) had to be coated. Cover glasses or plastic cover slips were placed in 4-well plates in

the laminar flow unit. Plastic culture dishes and 4-well plates were coated with poly-l-lysine,

dissolved in ultrapure water (100 µg/ml). To coat the culture dishes or 4-well plates, the

bottom of the slides was covered with a thin layer of poly-l-lysine, and placed in the incubator

overnight. The next day, slides were washed two times with DDW. Subsequently, 1st day

culture medium was added to the plates and placed in the incubator until use. Cover glasses

used in this study were not sterile. So they were autoclaved before use and stored under sterile

conditions.

3.3.14.2 Obtaining and cultivating primary murine neurons

All solutions were prepared in advance, adjusted to pH 7.4 and stored at 4° C.Hippocampal or

cortical cultures were obtained from the newborn mice (up to 24 h postnatally). All the

culturing procedure was performed on ice. Mice pups were decapitated and the brains were

dissected under sterile conditions. Brains were transferred into petri-dishes which contained

ice-cold dissection solution. Cortex and hippocampus were dissected and cleaned from blood

vessels and meninges. Subsequently, they were transferred into different petri-dishes, cut into

1 mm3 pieces and finally transferred into 15 ml tubes. After washing 4 times with 3 ml

dissection solution and treating with 5 ml Hanks solution, hippocampal and cortical slices

were warmed for 1 min in digestion solution, mixed with trypsin 3.3 mg/ml, DNAse 0.83

mg/ml, and treated with the same solution for 5 min at RT. The hippocampal and cortical

slices were thereafter incubated in dissection solution with 0.6 mg/ml trypsin inhibitor to

inhibit trypsin activity for 5 min and 3 min, respectively, and finally with fetal calf serum

(200 µl/ml in dissection solution) for 10 min. Hippocampal and cortical slices were washed

four times with dissection solution and homogenized in dissection solution, mixed with 0.4

mg/ml DNAse, with three different diameter of fire polished Pasteur pipettes. Five ml of

dissection solution was added and the cells were centrifuged (15 min; 800 x g; 4° C). To

determine the number of surviving cells in cell suspension, cells were counted following

trypan blue staining (see section 3.3.4). Subsequently, cells were plated on 4 well plates or

35 mm plastic culture dishes containing 1st day culture media (Culture Media, 10% horse

serum). After two days half of the culture media was replaced with 3rd day media and every

2nd-3rd day half of the media was replaced with fresh culture medium.


71

This culturing procedure resulted in a mixed neuronal culture containing 70-80% neurons.

The remaining cells were identified as astrocytes.

3.3.14.3 Treatment of primary cells

The JNK inhibitor SP600125 (2 µM) was given to the both cultures immediately after the

explantation and was applied with every change of culture medium.

3.3.14.4 Immunocytochemistry

Immunocytochemistry bases on the use of a primary antibody directed against the cellular

target(s) and a secondary antibody which is directed against the primary antibody and labeled

with an enzyme. The most commonly used enzymes are peroxidase and alkaline phosphatase.

Peroxidase activity is most frequently detected using 3,3’-Diaminobenzidine (DAB) as the

electron acceptor with hydrogen peroxide serving as the substrate. The reaction product forms

a brown precipitate at the site of the enzyme activity. To enhance the signal, the secondary

antibody is biotinylated and bound by avidin coupled to a complex containing the enzyme

(avidin-biotin-complex coupled with a peroxidase for the substrate reaction, i.e. the ABC

complex). Staining was performed using the following protocol.

Protocol:

1. Cells were incubated with pre-warmed para-formaldehyde (4% in PBS, 37° C) at RT

for 30 min

2. The fixed cells were permeabilized with Triton X-100 (0.2% in PBS) at RT for 2 min

3. Cells were then blocked in 5% normal goat serum, diluted in PBS

4. Cells were washed in 1% normal goat serum, diluted in PBS, two times for 3 min each

5. Cells were incubated with primary antibody (overnight at 4° C)

6. Cells were washed in PBS, three times for 3 min each

7. Cells were, hence, incubated with secondary antibody for 1 h (37° C)

8. Cells were washed in PBS, three times for 3 min each

9. Cells were incubated with ABC complex for 1 h. (37° C). Two drops of reagent A of

the Vectastain kit were added to 5 ml of PBS. After the addition of two drops of

reagent B the solution was mixed immediately. The solution was ready to use after

30 min.

10. Cells were washed in PBS, two times for 3 min each.


72

11. DAB solution was applied until staining was optimal as determined by light

microscopic examination (5-10 min at RT). One tablet of 3,3’-diaminobenzidine was

dissolved in 5 ml DDW. Each Sigma Fast DAB tablet contains DAB (0.7 mg/ml),

Urea Hydrogen Peroxide (0.2 mg/ml) and Tris buffer 0.06 M.

12. Cells were washed in DDW two times each for 5 min.

13. The stained cells were mounted and analyzed using a DMR microscope with a camera

system and the software LeicaQwin program.

3.3.15 Statistical analysis Data were analyzed using either GraphPad Prism or SPSS for Windows, version 14.0.

Kruskal-Wallis test was performed to verify that results from 3 - 6 experiments had no

significant differences before they were pooled to represent one joint control or treated group.

Data were subjected to Student’s t-test and one-way analysis of variance with repeated

measures and post-hoc Bonferroni analysis. For data that were not normally distributed

non-parametric statistical tests were run, consisting of Kruskal Wallis/Mann-Whitney U-test

and post-hoc Bonferroni or Tukey’s analysis.

P-value less than 0.05 was considered significant. All data are expressed as mean ± standard

deviation.

RESULTS

73

4. RESULTS

Table 21. Overview of all experiments.

Parameters investigated in Instrumental method

PC12 cells isolated mitochondria from PC12 (P) or mice brains (M)

hippocampal and cortical neurons

cell death ROS detection (M)

ROS detection change in ∆ΨM (M)

flow cytometry

change in ∆ΨM

spectro-fluorometry

change in ∆ΨM change in ∆ΨM (M)

ROS detection ROS detection (M) fluorescence plate reader

antioxidant potential of drugs

polarography oxygen consumption (M)

cytochrome c release

purity of mitochondria (M, P)

activation of mitochondrial JNK (P)

mitochondrial translocation (P)

inhibition of JNK translocation (P)

Western blot

JNK upstream kinases (P)

cytochrome c release

neurite length

subcellular JNK pools

effects of JNK inhibition

fluorescence microscopy

mitochondrial translocation of JNK

Abbreviations: JNK (c-Jun N-terminal kinases), ∆Ψm (mitochondrial membrane potential), ROS (reactive oxygen species)

RESULTS

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4.1 Mechanisms of 6-hydroxydopamine mediated cell death

4.1.1 6-hydroxydopamine induces cell death in PC12 cells

6-hydroxydopamine was administered at the concentration of 50 µM to the rat

pheocromocytoma cell line PC12. After 4 h (data not shown), 8, 12, 16 and 24 h cells were

analyzed by flow cytometry for markers of cell death (Annexin V-FITC and propidium

iodide) using the Becton Dickinson FACSCalibur flow cytometer. 6-OHDA stimulation lead

to a significant cell loss of about 28% ± 6% (P < 0.001) after 24 h (Figure 11A).

Furthermore, PC12 cells were treated for 24 h with 10, 25, 50, 100, or 200 µM 6-OHDA and

apoptosis/necrosis was assessed by flow cytometry. 6-OHDA promoted death among PC12

cells in a dose-dependent fashion. At 10 µM concentration 6-OHDA did not show a

significant decrease in the number of surviving cells, but cell death became evident at higher

concentrations: 25 µM caused 23% ± 4% loss of PC12 cells (P < 0.001), the use of 50 µM

promoted a loss of 31% ± 7% (P < 0.001), 100 µM and 200 µM showed a decrease by

49% ± 9% and 51% ± 8% (P < 0.001, each), respectively (Figure 11B). 6-OHDA induces cell

death in PC12 cells in a time- and dose-dependent manner.

4.1.2 6-hydroxydopamine generates reactive oxygen species in PC12 cells

In order to elucidate the mechanism of 6-OHDA mediated cell death the role of oxygen

radicals were investigated. ROS levels were assessed fluorometrically in the Fluoroskan

Ascent® plate reader by 2’,7’-dichlorofluorescein (DCF) fluorescence after administration of

the redox-sensitive dye 2’,7’-dichloro-dihydrofluorescein (H2-DCF). The values were

normalized according to the samples’ protein contents and results were displayed as

fluorescence intensity per mg protein in percent of control.

Incubation with 50 µM 6-OHDA was applied to investigate the time course of ROS

production. Stimulation periods were 4, 8, 12, 16, or 24 h. DCF fluorescence was 37% ± 8%

higher than controls after 4 h (P < 0.05), the earliest time point chosen (Figure 12A). The

minimal concentration to significantly generate ROS within 4 h was 25 µM (Figure 12B).

Levels were elevated by 18% ± 3% (P < 0.05).

RESULTS

75

Figure 11. 6-hydroxydopamine and cell death

PC12 cell death was assessed using Annexin V-FITC/PI in the flow cytometer. From the original dot plots viable cells were identified, total number of surviving cells was counted and compared to controls. A) Time course of 6-OHDA mediated cell death at a concentration of 50 µM. There is a significant decrease in the number of viable PC12 cells after 24 h. B) Concentration-dependent decrease in viability 24 h after inbubation with 6-OHDA. Vehicle-treated controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), FI (fluorescence intensity), FITC (fluorescein-isothiocyanate), PI (propidium iodide). *** P < 0.001 Statistical significances were calculated using t-test. Data are presented as mean ± standard deviation. N = 3.

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Figure 12. Reactive oxygen species levels after 6-OHDA

Reactive oxygen species (ROS) levels were measured fluorometrically in PC12 cells after 6-OHDA and incubation with H2DCF. A) Time course of ROS production following 50 µM 6-OHDA shows an early increase in DCF fluorescence after 4 h. B) 50 µM 6-OHDA and higher concentrations induce ROS production in PC12 cells within 4 h of treatment. Vehicle-treated controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), DCF (dichlorofluorescein), FI (fluorescence intensity), ROS (reactive oxygen species). * P < 0.05; ** P < 0.01; *** P < 0.001 Statistical significances were calculated using t-test. Data are presented as mean ± standard deviation. N = 6.

RESULTS

77

These findings raised the need to conduct additional experiments to assess early effects of

ROS generation by 6-OHDA. 50 µM 6-OHDA was administered for 15 min, 30 min, 1 h and

2 h and DCF fluorescence was measured from samples in the fluorescence plate reader and by

flow cytometry. Both methods revealed that the very first significant increase in fluorescence

intensity occured after 60 min of stimulation (Figure 13). Staurosporine (STS) was used as a

positive control for increased ROS production at a concentration of 1 µM.

Given the profound changes of ROS levels in early stages of 6-OHDA intoxification, we were

curious to assess the source of ROS in this model. H2DCF is a useful tool for monitoring ROS

generated in the cytosol or mitochondrial intermembrane space, and ROS that are released

from mitochondria (Diaz et al., 2003). Among the redox-sensitive fluorescent dyes dihydro-

rhodamine (DHR) is most sensitive towards oxidation by hydrogen peroxide (Walrand et al.,

2003), especially in the presence of cytochrome c oxidase in mitochondria (Royall and

Ischiropoulos, 1993). Thus, it can be used as a specific marker for the detection of

mitochondrial ROS.

ROS levels as measured by DCF and DHR fluorescence in the flow cytometer after 60 min

incubation with 6-OHDA were increased similarly in the dot plots (Figure 14A). Regions of

interest (ROI) were drawn in the control dot plots in a manner that any higher fluorescence

intensity for DCF or DHR would appear in this region and therefore become available for

counting with the CellQuest program. After 60 min stimulation with 50 µM 6-OHDA almost

the whole cell populations appeared in the ROIs (R3 for DCF, R4 for DHR), shifting upwards

due to higher fluorescence intensity in the samples. DCF fluorescence intensity increased

from 4% ± 3% to 90% ± 3%, whereas DHR fluorescence increased from 8% ± 2% in control

cells to 81% ± 2% after 6-OHDA. Based on the assumption that DHR is oxidized by a

subpopulation of ROS that can be detected by DCF, a 90% overlap of mitochondrially

generated ROS (as measured by DHR) and whole-cell DCF fluorescence was calculated

(Figures 14B and 14C).

Oxidative stress is a very early event occuring within 60 min following the administration of

6-OHDA to PC12 cells. The source of ROS largely appears to be mitochondria.

RESULTS

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Figure 13. Reactive oxygen species at early time points

Reactive oxygen species (ROS) levels were measured by DCF fluorescence in PC12 cells after 6-OHDA. There was a critical increase when 6-OHDA was given for one hour. A) Histogram depicting ROS production in PC12 cells after 50 µM 6-OHDA as measured by flow cytometry. B) Bar chart showing FI changes in percent of control in PC12 cells following 50 µM 6-OHDA and 1 µM staurosporine (STS), which served as a positive control. Vehicle-treated negative controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), DCF (dichlorofluorescein), FI (fluorescence intensity), ROS (reactive oxygen species), STS (staurosporine) * P < 0.05; ** P < 0.01; *** P < 0.001 Statistical significances were calculated using t-test. Data are presented as mean ± standard deviation. N = 3.

RESULTS

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Figure 14. Sources of reactive oxygen species

Reactive oxygen species (ROS) levels were measured by DCF and DHR fluorescence intensity (FI) in PC12 cells after 1 h of incubation with 50 µM 6-OHDA. A) Dot plots depicting control cell populations and PC12 cells after stimulation. B) Quantitative analysis of DCF and DHR fluorescence. Bar charts represent the number of events in the ROIs (R3 for DCF, R4 for DHR) for control cells (left columns) and 6-OHDA-treated samples (right columns). C) DHR FI values are 90% of the increase of DCF FI after 1 h 6-OHDA. Vehicle-treated controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), DCF (dichlorofluorescein), DHR (dihydrorhodamine), FI (fluorescence intensity), ROS (reactive oxygen species). *** P < 0.001 Statistical significances were calculated using t-test. Data are presented as mean ± standard deviation. N = 3.

RESULTS

80

4.1.3 The mitochondrial membrane potential collapses after 6-hydroxydopamine

treatment

ROS in mitochondria are originating from defects in the respiratory chain. Under healthy

conditions there is a constant low-level ROS generation due to proton leakage. Following a

disruption of the mitochondrial membrane potential, i.e. during apoptosis, ROS production

increases exponentially. Therefore the question aroused if the marked elevation of ROS levels

is due to the disruption of the mitochondrial membrane potential. The dual-fluorescent probe

JC-1 is a tool to analyze the mitochondrial membrane potential (∆Ψm) in situ. Emission at 530

nm (green) derives from JC-1 monomers whereas aggregates emit fluorescence at 590 nm

(red). Those aggregates form preferably in slightly acidic environment as it is found in the

mitochondrial matrix due to the activity of the electron transport chain. Consequently,

J-aggregates are regarded to be a sensor for mitochondrial respiration and mark an intact

mitochondrial membrane potential (Reers et al., 1991; Smiley et al., 1991; Cossariza et al.,

1993). When the mitochondrial membrane potential gets disrupted, aggregates dissociate and

the fluorescence intensity (FI) of monomers rises.

First, preliminary tests were performed to analyze how healthy PC12 cells react to different

concentrations of JC-1, and which concentration to choose best. Concentrations of 0.1 µM,

1 µM, 5 µM, 10 µM and 50 µM JC-1 were applied to PC12 cells and the fluorescence

intensities from the suspensions were measured fluorometrically in the cuvette. Fluorescence

intensity of the aggregates at 590 nm was generally higher than compared to that of the

monomers (530 nm). The emission in the red range was clearly dose-dependent (Figure 15A),

whereas the fluorescence in the green range displayed the pecularity that 5 µM and 10 µM

JC-1 resulted in similar curves (Figure 15B). Conclusively, 10 µM JC-1 were chosen for

further experiments, since this concentration provided the optimal aggregate/monomer ratio

regarding healthy PC12 cells.

The next step was to analyze the stability of JC-1 fluorescence over the experimental time.

The responsiveness of the model was verified using valinomycin as a positive control, an

agent that acts as an ionophore and depolarizes the mitochondrial membrane potential. 1 µM

was administered at the end of each 90 min session. As for the samples, untreated PC12 cells

were selected, but also solvents that are contained in the stock solutions of 6-OHDA,

valinomycin, and JC-1 itself (ascorbic acid, ethanol and DMSO) were checked. Finally, the

importance of keeping the samples on ice during the experiment was evaluated. Dot plots

RESULTS

81

from flow cytometrical analysis were recorded and a region of interest (ROI) was selected to

display events with high red and low green fluorescence, indicative for healthy cells. The

change of the number of events in this ROI was analyzed.

Figure 15. JC-1 dual fluorescence in healthy PC12 cells

Different concentrations of JC-1 and their fluorescence intensities (FI) in untreated PC12 cells followed for 1400 seconds. FI was measured fluorometrically in the cuvette by adding the dye to comparably concentrated cell suspensions. A) JC-1 emission at 590 nm (red). B) Fluorescence emission at 530 nm (green). 10 µM JC-1 provides the optimal ratio to describe ∆Ψm in healthy PC12 cells. Exemplary results (N=3) are displayed. Abbreviations: FI (fluorescence intensity), ∆Ψm (mitochondrial membrane potential)

JC-1

RESULTS

82

Untreated PC12 cells, as well as PC12 cells treated with ascorbic acid, ethanol or DMSO,

maintained their ∆ΨM over a time course of 90 min (Figure 16). Untreated PC12 cells

responded to 1 µM valinomycin with a 72% decrease of red fluorescent events. Ascorbic acid,

which was added to 6-OHDA stocks in a concentration of 0.02%, apparently attenuated

depolarizing effects (62% decrease) under the same conditions as untreated cells, but the

difference was not significant compared to untreated cells. J-aggregate FI decreased by 76%

and 83% after addition of ethanol alone (used in JC-1 stocks at a concentration of 0.1%) and

DMSO, respectively (final concentration 0.1%). The combination of all solvents in the

analysis medium as applied to “control cells“ led to a similar decline (83%), despite the

presence of ascorbic acid.

In all samples the FI of the 90 min time point is obviously lower than the previous values,

therefore the assumption was made that the mitochondrial membrane could loose integrity

after keeping PC12 in the vial for longer than 90 min. Handling the samples on ice could most

likely prolong this time window, since the strongest and most consistent FI was found in this

group. Still, cells were not handled on ice, because the response to valinomycin was weak

(only 16% decrease of red fluorescence; P < 0.001) and could also affect the results from

treatment with 6-OHDA (Figure 16). For further experiments samples were not kept on ice,

since the responsiveness to the depolarizing agent valinomycin was obviously handicapped

and could mask effects of 6-OHDA when applied.

RESULTS

83

Figure 16. Stability of JC-1 fluorescence over the experimental time (see next page)

solvent

1 µM Valinomycin

RESULTS

84

Figure 16. Stability of JC-1 fluorescence over the experimental time (see previous page)

Graphs show the number of events calculated from a region of interest (ROI) representing an intact mitochondrial membrane potential (∆ΨM). The intact ∆Ψm is demonstrated by low green and high red fluorescence intensity (FI) based on dot plots acquired by flow cytometry in percent of total number of events. A) Panel shows the change of events in the ROI after a follow-up time of 90 min and the response to 1 µM valinomycin after the observation period. The effect of different solvents on ∆ΨM was measured as well as stability over time with or without keeping the samples on ice meanwhile. B) Bars show the average mean of events in the ROI over the investigation period and the effect of 1 µM Valinomycin on ∆ΨM. Abbreviations: ROI (region of interest), ∆Ψm (mitochondrial membrane potential), FI (fluorescence intensity), DMSO (dimethylsulfoxide), Val. (valinomycin) * P < 0.05; *** P < 0.001 Statistical significances were calculated using one-way ANOVA with post-hoc Bonferroni. Data are presented as mean ± standard deviation. N = 3.

As a next step the effect of 6-OHDA at concentrations of 10µM, 25 µM, 50 µM or 100 µM on

∆ΨM was investigated in PC12 cells at various incubation times (4, 8, 12, 16, or 24 h) and

fluorescence of J-aggregates (emission at 590 nm) was assessed in the flow cytometer.

Analysis was based on the number of events in a region of interest that contained the vast

majority of events in healthy controls (upper left quadrant in the dot plot as brought in

Figure 17).

A decrease in the red fluorescence counts specifically for a depolarization of the

mitochondrial membrane (Bernardi et al., 1999). After 8 h red fluorescence intensities (FI) of

samples incubated with 25 µM, 50 µM and 100 µM 6-OHDA decreased substantially

(Figures 17 and 18). 25 µM account for values of 80% ± 6% of those of controls (P < 0.05),

stimulation with 50 µM or 100 µM resulted in a decrease down to 65 ± 10% or 62% ± 6%,

respectively (P < 0.001). Further duration of treatment resulted in continously low levels of FI

at 590 nm after 50 µM or 100 µM 6-OHDA (61% ± 5% or 47% ± 6% of control levels;

P < 0.001) (Figure 18A). Upon uncoupling the respiratory chain with 1 µM valinomycin the

mitochondrial membrane became completely depolarized. The FI at 590 nm was 36% in

average following treatment with these agents. Taking into account this range between

controls and complete ∆ΨM disruption by valinomycin, 25 µM, 50 µM and 100 µM

depolarized the mitochondrial membrane by 32% ± 9%, 55% ± 16% and 59% ± 10%,

respectively, after 8 h. At the 12 h time point, the decrease was 61% ± 7% and 84% ± 9%

after 50 µM and 100 µM 6-OHDA, respectively.

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85

Figure 17. Time point of disruption of the mitochondrial membrane potential

Figure depicts exemplary results from 3-5 flow cytometrical analyses of PC12 cells following 50 µM of 6-OHDA. A) Graph presenting a time-dependent shift in the red fluorescence with a distinct decrease after 8 h. B) Dot plot analysis showing a control cell population with distinct intensities of red and green fluorescence. C) Dot plot analysis of PC12 cells treated with 50 µM 6-OHDA for 8 h. The cell population shifts to a segment of higher green and lower red fluorescence. D) and E) Valinomycin disrupts the ∆Ψm. 1 µM served as a positive control depolarizing the mitochondrial membrane in PC12 cells as shown by the population shift to the lower right. Vehicle-treated controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), ∆Ψm (mitochondrial membrane potential), FI (fluorescence intensity)

RESULTS

86

A different way to present data from JC-1 fluorescence is the determination of the ratio

between red and green fluoresence. Although it has been found that only the decrease in red

fluorescence is specific for the disruption of ∆ΨM, this method is still widely used. Less red

and stronger green fluorescence result in smaller ratios, indicating a disruption of ∆ΨM. The

number of events positive for JC-1 fluorescence at 590 nm (cells in the upper left quadrant)

was divided by the number of events exhibiting high green fluorescence intensity (upper right

and lower right quadrant). Ratios decreased over time following 6-OHDA in different

concentrations, with, generally, the greatest decrease between 4 h and 8 h of incubation.

50 µM and 100 µM, however, exerted the lowest ratios (Figure 18B). Again, 1 µM

valinomycin was applied to assess what values would represent a totally depolarized

mitochondrial membrane. The JC-1 aggregate/monomer ratio acquired by this procedure was

approx. 1.2. Thus, 10 µM 6-OHDA caused depolarization by 10% (4 h), 60% (8 h) and 54%

(12 h); 25 µM yielded 29% (4 h) and 69% (8 h, P < 0.05; and 12 h); 50 µM showed a 33%

(4 h), 91% (8 h, P < 0.001) and 95% (12 h, P < 0.001) decrease whereas 100 µM 6-OHDA

resulted in a drop of 59% (4 h), 94% (8 h, P < 0.001) and 105% (12 h, P < 0.001) of ∆ΨM

(Figure 18B).

Immediate effects following the administration of 6-OHDA were analyzed fluorometrically in

a time-resolved manner. JC-1 red (excitation wavelength 488, emission wavelength 590) and

green fluorescence (488:530) from a PC12 cell suspension in a cuvette, followed over a time

period of 2000 seconds. At the indicated time, 50 µM 6-OHDA were pipetted into the

solution, followed by a 30 seconds break before continuous measurement to reduce effects of

turbulences. The FI in both emission wavelengths dropped, although changes in the green

fluorescence were weak, and subsequently the red fluorescence decreased at a steeper slope

than the base line. The ratio graph endorsed the immediate decline of ∆ΨM (Figure 19).

Following concentrations over 25µM 6-OHDA PC12 cell mitochondria loose their integrity

as shown by the disruption of ∆ΨM after 8 h. Not the time point but the initial concentration of

the toxin seems to be the crossroads for milder ∆ΨM disturbance or heavy disruption of the

electrochemical gradient.

RESULTS

87

Figure 18. Mitochondrial membrane potential in PC12 cells following 6-OHDA

PC12 cells were incubated with 10 µM JC-1 and fluorescence intensity (FI) was measured by flow cytometry as described. A) 25 µM, 50 µM and 100 µM reduced the number of red fluorescent cells significantly after 8 h of incubation with 6-OHDA. B) Ratio of red/green fluorescence at the investigated time points. Vehicle-treated controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), ∆ΨM (mitochondrial membrane potential), FI (fluorescence intensity) * P < 0.05; *** P < 0.001 Statistical significances were calculated using one-way ANOVA with post-hoc Bonferroni. Data are presented as mean ± standard deviation. N = 4.

RESULTS

88

Figure 19. Immediate effect of 6-OHDA on ∆ΨM of PC12 cells

JC-1 red (excitation wavelength 488, emission wavelength 590) and green fluorescence (488:530) from a PC12 cell suspension (large graph) and their ratio (red vs. green; graph in the upper right corner), followed over a time period of 2000 seconds. JC-1 was used at a concentration of 10 µM. 50 µM 6-OHDA were given at the time point marked. The asymptote before 6-OHDA marks the base line, the second line’s slope indicates an ongoing disturbance of ∆ΨM resulting from the administration of the toxin. Abbreviations: 6-OHDA (6-hydroxydopamine), FI (fluorescence intensity), ∆Ψm (mitochondrial membrane potential)

6-OHDA

6-OHDA

RESULTS

89

4.1.4 6-OHDA induces cytochrome c release

JC-1 shows the impairment of the electron transport chain of mitochondria and ∆ΨM

disruption which leads to formation of permeability transition pores, release of mitochondrial

proteins and caspase activation leading to cell death (Cossariza et al., 1993). The

mitochondrial death pathway involves the release of cytochrome c into the cytosol. Under

basal conditions cytochrome c is situated in the mitochondrial intermembrane space, mainly

attached to the inner mitochondrial membrane, and is only found in the cytosol when the

mitochondrial membrane integrity is lost. Substantial levels of cytochrome c were identified

in cytosolic PC12 cells fractions after 8 h incubation with 25 µM 6-OHDA, as demonstrated

by Western blot analysis (Figure 20A). Findings were confirmed using fluorescence

microscopy of methanol/paraformaldehyde-fixed samples stained with anti-cytochrome c

antibody and FITC-conjugated secondary antibody. In control cells cytochrome c staining is

clustered (hint for localization in mitochondria), whereas 8 h after 25 µM 6-OHDA the

distribution is changed to a completetely diffuse pattern (cytochrome c in the cytosol)

(Figure 20B).

Figure 20. Cytochrome c release

Detection of cytochrome c release into the cytosol. Displayed are representative results (n = 3). A) In the Western blot substantial levels of cytochrome c were identified in the cytosolic fraction of PC12 cells after 8 h. B) Fluorescence microscopy of cells stained with anti-cytochrome c antibody combined with FITC-conjugated secondary antibody. Cytochrome c distribution changes from clusters (mitochondria) to a diffuse pattern (cytosol) after 8 h incubation with 25 µM 6-OHDA. Vehicle-treated controls were used. Scalebar represents 20 µm.

16.5 kDa

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4.2 Responsiveness of isolated mitochondria to 6-hydroxydopamine

4.2.1 Analyses with isolated mitochondria

The next experiments addressed the question if isolated mitochondria responded to 6-OHDA

in similar manner as cells. Isolated mice brain mitochondria were analyzed flow

cytometrically. The production of reactive oxygen species (ROS) was determined using ROS-

sensitive fluorescent dyes, and the integrity of the mitochondrial membrane was assessed by

the dual-fluorescent dye JC-1. Additionally, the immediate effect of 6-OHDA on the

mitochondrial membrane potential of a mitochondrial suspension in the cuvette was measured

fluorometrically in a time-resolved manner.

4.2.2 Quality of isolated mitochondria

Subsequent to every isolation, a sample of mitochondria was studied for their functional

integrity by assessing the RCR (Figure 21). Respiration on succinate (feeding complex II) in

the presence of rotenone (inhibiting complex I) was established and ADP was added. State 4

respiration was calculated as the slope (m = ∆y/∆x) of the asymptote, as it was done for

state 3 respiration, after all ADP had been used up. In the example brought in Figure 21, the

RCR was calculated as 0.5/0.16 which resulted in 3.125. Mitochondria with a RCR above 3

were acceptable for use in experiments and were stable for the next 2 - 3 h.

Figure 21. Assessment of mitochondrial functional integrity

Isolated mitochondria were analyzed for their functional integrity by measuring oxygen consumption with a Clark electrode. The slopes of state 3 and state 4 respiration are drawn. The RCR of this example was calculated as 3.125. Abbreviations: ADP (adenosin-diphosphate), [O] (concentration of molecular oxygen in the sample), RCR (respiratory control ratio), Rot. (rotenone), Suc. (succinate).

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4.2.3 Purity of mitochondrial fractions

Cytosolic and mitochondrial fractions obtained during the isolation procedure were subjected

to Western blot analysis. Membranes were incubated with antibodies against the

mitochondrial constitutional protein cytochrome c oxidase subunit IV (COX IV) and ß-actin

for the assessment of cytosolic contaminations. Usually, ß-actin was present in low-level

concentrations even in highly purified mitochondria (Figure 22). This can be explained by the

tight connections between mitochondria and the cytoskeleton together with the nature of the

isolation method.

Figure 22. Purity of mitochondrial fraction obtained from mice brain

Representative Western blot detecting protein levels of ß-actin (cytosolic marker) and cytochrome c oxidase subunit IV (COX IV; mitochondrial marker). The mitochondrial fraction (mito) shows high COX IV expression and little amounts of ß-actin. Abbreviations: COX IV (cytochrome c oxidase subunit IV), mito (mitochondrial fraction).

4.2.4 ROS levels in isolated mitochondria following 6-OHDA

Samples of isolated mitochondria were preloaded with 2’,7’-dichloro-dihydrofluorescein

(H2DCF) and incubated with 6-OHDA. Concentrations of 10, 25, 50, or 100 µM 6-OHDA

were not sufficient to increase ROS levels in isolated mitochondria significantly when

administered for 60 min, as measured in the Fluoroskan plate reader (Figure 23). Only at a

very high concentration (200 µM) a significant increase of DCF FI indicated higher ROS

levels. DCF was sufficient as a marker of ROS in mitochondrial preparations since it can pass

the outer mitochondrial membrane and therefore indentify ROS generated in the

mitochondrial intermembrane space. Secondly it would be oxidized by ROS diffusing from

the matrix.

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4.2.5 The mitochondrial membrane potential of isolated mitochondria following

6-OHDA

The mitochondrial membrane potential was measured with the aid of the dual-fluorescent

redox sensitive probe JC-1. Emission at 530 nm (green) derives from the monomers whereas

aggregates, which are formed in the slightly acidic milieu of the mitochondrial matrix, emit

fluorescence at 590 nm (red). When the mitochondrial membrane potential gets disrupted,

aggregates dissociate and the fluorescence intensity (FI) of monomers rises. Changes in ∆ΨM

upon various concentrations of 6-OHDA treatment (10, 25, 50, and 100 µM) was measured

by JC-1 fluorescence in the BD FACSCalibur flow cytometer. Administration of 50 µM

6-OHDA resulted in an increased FI of monomers (530 nm) while red FI (590 nm) decreased

at the same time indicating a disruption of ∆ΨM (Figure 24A and B).

Figure 23. Measurement of reactive oxygen species in mitochondrial fractions

Reactive oxygen species (ROS) levels were measured by DCF fluorescence in 24 well plates in mitochondria isolated from mice brain. 200 µM 6-OHDA applied for 60 min elevated ROS levels significantly, altogether an increase in DCF fluorescence could be observed. Vehicle-treated controls were used. Abbreviations: FI (fluorescence intensity), DCF (dichlorofluorescein), ROS (reactive oxygen species). *** P < 0.001 Statistical significances were calculated using t-test. Data are presented as mean ± standard deviation. N = 3.

Presenting the mitochondrial population in a dot plot (530 nm versus 590 nm) reveals an

evident susceptibility of isolated mitochondria to 6-OHDA (30 min incubation), as shown by

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the shift into the lower right quadrant (Figure 24C and D). Quantitative analysis of the

number of events in the upper left quadrant (Figure 24C and D), representing the healthy

mitochondrial population, was performed. The red fluorescence of J-aggregates decreased

significantly (58% of control levels) after 30 min when 100 µM 6-OHDA were applied

(P < 0.05). 40 min stimulation with 50 µM or 100 µM 6-OHDA disrupted ∆ΨM by 51% each

(P < 0.05, and P < 0.01, respectively). After 50 min, FI at 590 nm decreased significantly at

all concentrations. Incubation with 10 µM resulted in FI of 66% of control levels (P < 0.05),

the other values were 60% following 25 µM (P < 0.05) and 41% following 50 µM or 100 µM

(P < 0.01). 60 min stimulation with 10 µM 6-OHDA decreased ∆ΨM to 63% of control levels

(P < 0.05), 25 µM led to 55% (P < 0.05), ∆ΨM decreased to 41% following 50 µM (P < 0.01)

and 31% (P < 0.001) after treatment with 100 µM 6-OHDA (Figure 25A). Full depolarization

of ∆ΨM with 1 µM valinomycin gave a ratio of approx. 0.9. The administration of 10 µM

6-OHDA caused, hence, a depolarization by 85% (50 min, P < 0.05) and 88% (60 min,

P < 0.05). 25 µM resulted in a decrease of 90% (50 min, P < 0.05) and 95% (60 min,

P < 0.05). 50 µM showed a 94% decline of ∆ΨM after 40 min (P < 0.05), and a drop of 102%

(50 and 60 min, P < 0.01), whereas 100 µM 6-OHDA decreased ∆ΨM by 91% (30 min,

P < 0.05), 99% (40 min, P < 0.01), 105% (50 min, P < 0.01) and 110% (60 min, P < 0.001)

(Figure 25B).

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Figure 24. Isolated mitochondria and the mitochondrial membrane potential

Depicted are results from flow cytometrical analysis of ∆ΨM of isolated mice brain mitochondria. A) Curves show a time-dependent increase of mitochondria emitting green fluorescence (530 nm) after 50 µM 6-OHDA. B) Accordingly, there is a decrease in the red fluorescence upon longer incubation with 6-OHDA. C) Dot plot presents a representative example of a control mitochondrial population, with higher FI for the red than for the green emitting wavelength. A quadrant was set for later statistical analysis. D) After 100 µM 6-OHDA (30 min) the mitochondrial population shifts into the lower right quadrant, indicative for ∆ΨM disruption. Vehicle-treated controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), FI (fluorescence intensity), ∆ΨM (mitochondrial membrane potential)

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Figure 25. Mitochondrial membrane potential in isolated mitochondria following

6-hydroxydopamine

Isolated mitochondria were preloaded with 10 µM JC-1, then stimulated with 6-OHDA, and fluorescence intensity (FI) was measured by flow cytometry. Analysis is based on the number of events in a region of interest that was set to contain the vast majority of events in healthy controls (upper left quadrant in the dot plot as depicted in Figure 19C). A) Effects of various concentrations of 6-OHDA on ∆ΨM of isolated mitochondria. B) Ratio of red/green fluorescence at the investigated time points. Vehicle-treated controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), ∆ΨM (mitochondrial membrane potential), FI (fluorescence intensity) * P < 0.05, ** P < 0.01, *** P < 0.001 Statistical significances were calculated using one-way ANOVA with post-hoc Bonferroni. Data are presented as mean ± standard deviation. N = 4.

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Suspensions of isolated brain mitochondria were diluted 1:20 and subjected to fluorometrical

analysis in a time-resolved manner to discover immediate changes in ∆ΨM following

6-OHDA. JC-1 red (ex. 488, em. 590) and green fluorescence (ex. 488, em. 530) were

followed over a time period of 1600 sec. Before the addition of 6-OHDA isolated

mitochondria were administered rotenone (10 µM) and succinate (0.5 mM), rotenone

inhibiting complex I while succinate being the substrate to feed complex II of the respiratory

chain. After achieving stable conditions, 10 µM 6-OHDA was pipetted into the solution,

followed by a 30 seconds break before continuous measurement to reduce effects of

turbulences. The result was an immediate decrease of mainly the red signal, and the ratio of

red to green fluorescence (Figure 26), indicating a loss of mitochondrial membrane integrity.

Figure 26. Immediate effect of 6-OHDA on isolated mitochondria

The representative graph shows JC-1 red (ex. 488, em. 590) and green fluorescence (ex. 488, em. 530) from a suspension of isolated mitochondria, followed fluorometrically over a time period of 1600 seconds. Before the addition of 6-OHDA isolated mitochondria were administered rotenone (10 µM) and succinate (0.5 mM). At the indicated time, 10 µM 6-OHDA was pipetted into the solution. The graph in the upper right corner depicts changes in the ratio of red to green fluorescence over the duration of the experiment. Administration of 1 µM Valinomycin resulted in a further drop of the ratio. Abbreviations: 6-OH (6-OHDA), BM (brain mitochondria), FI (fluorescence intensity), Rot./R (rotenone), Succ./S (succinate), Val./V (valinomycin)

Val.

6-OHDARot. Succ. R S 6-OH V

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4.3 The mitochondrial death pathway and c-Jun N-terminal kinase

signaling

c-Jun N-terminal kinases (JNK) are central mediators in stress-signaling pathways and play a

role in the mitochondrial death pathway. Kharbanda et al. found that JNK translocated to

mitochondria in various cell lines following ionizing radiation (Kharbanda et al., 2000). We

hypothesized that 6-OHDA treatment could provoke JNK translocation to mitochondria as

well.

4.3.1 Purification of PC12 cell mitochondrial fractions

At first, an isolation method to obtain mitochondrial fractions from PC12 cells was

established. The purity of these fractions was analyzed by Western blot detection of

mitochondrial and cytosolic markers (Figure 27). Lysed PC12 cells (C), intermediate samples

from the isolation procedure (I1, I2; see materials and methods, section 3.3.5) and the final

mitochondrial fraction were stained with antibodies against the mitochondrial resident protein

cytochrome c oxygenase subunit IV (COX IV) and ß-actin, to determine cytosolic

contaminations of the fractions. It was revealed that the first mitochondrial fraction obtained

during the isolation procedure (I1) was already rich in mitochondrial COX IV protein, and

contained rather low levels of ß-actin, but the washing step reduced cytosolic contamination

further (I2). The final mitochondrial fraction (M) was almost cleared from ß-actin, and was

enriched in mitochondrial COX IV protein.

Figure 27. Isolation and purification of mitochondria from PC12 cells

Western blot analysis of representative intermediate samples from the isolation procedure to obtain mitochondrial fractions from PC12 cells. Expression of cytosolic ß-actin and mitochondrial cytochrome c oxidase subunit IV (COX IV) was determined for samples taken before the incubation in sucrose buffer (controls, C), two samples from in between the centrifugation steps (I1 and I2), and one taken from the final mitochondrial suspension (M). Abbreviations: C (controls), COX IV (cytochrome c oxidase subunit IV), I1, I2, M (see legend text).

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4.3.2 Activation of the mitochondrial pool of JNK following 6-OHDA

Mitochondrial fractions from PC12 cells were analyzed by Western blot for their total JNK

and phospho- (active) JNK content after incubation with 25 µM 6-OHDA for various

durations (4, 8, 12, 16, 24, and 48 h) (Figure 28). Total-JNK protein content is virtually

unchanged regardless of the stimulation. Under basal conditions, hardly any phosphorylated

JNK could be detected in mitochondrial fractions. Phospho-JNK expression is upregulated

within 4 h and levels remain high up to 24 h after 6-OHDA addition (Figure 28).

Figure 28. Activation of JNK in mitochondrial fractions

Representative results of Western blot analysis of total- and (active) phospho-JNK levels in mitochondrial fractions of unstimulated PC12 cells and following incubation with 25 µM 6-OHDA for 4, 8, 12, 16, 24 and 48 h. Grp75 is a residential protein in the mitochondrial matrix. Vehicle-treated cells served as controls. Abbreviations: 6-OHDA (6-hydroxydopamine), con (unstimulated control), JNK (c-Jun N-terminal kinase)

4.3.3 Translocation of c-Jun N-terminal kinase isoform 2 (JNK2) to mitochondria

Detection of JNK isoforms 1 and 2 in Western blots of mitochondrial preparations revealed

differential patterns. The pool of JNK1 at the mitochondria did not change after addition of

25 µM 6-OHDA. In contrast, JNK2 levels in basal conditions were low and increased upon

stimulation in mitochondrial fractions within 4 h (Figure 29A). The JNK inhibitor SP600125

was able to prevent JNK2 translocation as compared to stimulated cells at the four hour time

point, but had no effect on the presence of JNK1 at the mitochondria (Figure 29B).

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Figure 29. JNK1 and JNK2 expression in mitochondrial fractions

Representative results of Western blot analysis of mitochondrial fractions from PC12 cells. Grp75 is a residential protein in the mitochondrial matrix. A) JNK1 and JNK2 expression in mitochondrial fractions of unstimulated controls and following 1 h, 2 h, and 4 h incubation with 25 µM 6-OHDA. B) JNK1 and JNK2 expression in mitochondrial fractions of unstimulated controls, and after 4 h stimulation with 25 µM 6-OHDA, with or without the JNK inhibitor SP600125 (SP). Vehicle-treated controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), con (unstimulated control), SP (SP600125), JNK (c-Jun N-terminal kinase)

To support these findings, confocal microscopy was performed with samples of PC12 cells

stained with mitotracker red, anti-JNK primary antibodies plus FITC-conjugated secondary

antibodies and Hoechst 33258 for visualization of nuclei. JNK1 distribution in controls

showed some overlay with mitochondria in the merged picture, but generally JNK1 was found

throughout the cytosol, and particulately in the nucleus. Treatment with 50 µM 6-OHDA did

not change the pattern apart from slightly more clustering and a minor increase of JNK1 in the

nucleus (Figure 30).

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Figure 30. JNK1 distribution in PC12 as measured by confocal microscopy

In preparation for confocal microscopy, nuclei were stained with the DNA dye Hoechst 33258, mitochondria were localized by mitotracker red (MTred) and JNK1 was visualized with anti-JNK1 primary antibody plus FITC-conjugated secondary antibody. JNK1 expression in control cells was mainly cytosolic, with some hints for basal expression in mitochondria and nuclei. Following 6-OHDA (50 µM, 4 h), a slight increase of JNK1 levels in the nucleus could be detected in the merged picture. Scalebar represents 20 µM. Abbreviations: 6-OHDA (6-hydroxydopamine), JNK (c-Jun N-terminal kinase), MTred (mitotracker red), FITC (fluorescein-isothiocyanate)

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Figure 31. JNK2 distribution in PC12 as measured by confocal microscopy

In preparation for confocal microscopy, nuclei were stained with Hoechst 33258, mitochondria were localized by mitotracker red (MTred) and JNK2 was visualized with anti-JNK2 primary antibody plus FITC-conjugated secondary antibody. JNK2 expression in control cells was low and appeared to be cytosolic. Following 6-OHDA (50 µM, 4 h), JNK2 was found to be clustered and co-localized with mitochondria, as brought in the merged picture. Scalebar represents 10 µM (for control) and 20 µM (for 6-OHDA), respectively. Abbreviations: 6-OHDA (6-hydroxydopamine), JNK (c-Jun N-terminal kinase), MTred (mitotracker red), FITC (fluorescein-isothiocyanate)

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JNK2 expression was weaker in controls as compared to JNK1 levels and displayed no

co-localization with mitochondria (Figure 31). Stimulation with 50 µM 6-OHDA resulted in

bright clusters of JNK2 at the same spots as mitochondria were stained confirming near

proximity/co-localization of both (Figure 31). Levels in the nucleus were a bit higher than in

controls, but not as high as JNK1 levels were under the same conditions.

4.3.4 Upstream kinases and JNK scaffolds in mitochondrial fractions

The upstream kinases for JNK comprise MKK4 and MKK7. To differentiate their

contribution to the translocation of JNK2 to mitochondria, the expression of these MAPKKs

was analyzed by Western blot. MKK4 was present in mitochondrial preparations from PC12

cells, and levels did not change following 25 µM 6-OHDA. Nonetheless, the pool of MKK4

was activated by addition of an active phoshpate 2 - 4 h after 6-OHDA stimulation

(Figure 32A). Administration of SP600125 attenuated the presence of MKK4 in

mitochondrial fractions compared to 4 h treatment with 6-OHDA alone (Figure 32B). MKK7

was not found in mitochondrial fractions at all, control experiments confirmed MMK7

expression in the cytosol (Figure 32C). The JNK scaffold JIP was present at mitochondria,

but expression levels remained unchanged following stimulation with 6-OHDA (Figure 32D).

Taken together, 6-OHDA induced the translocation of MKK4 and the JNK isoform 2 to

mitochondria, as well as the activation of these kinases within 4 h. Subsequently, 6-OHDA

treatment resulted in the release of cytochrome c from the mitochondrial intermembrane space

into the cytosol, which is a key event in apoptotic processes.

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Figure 32. Upstream kinases and scaffolds in mitochondrial fractions

Representative results of Western blot analysis. A) MKK4 was found in mitochondrial preparations from PC12 cells, its levels did not change following 25 µM 6-OHDA up to 4 h. However, 6-OHDA activated mitochondrial MKK4 between 2 h and 4 h. B) Expression of phospho-MKK4, but not MKK4, was reduced below control levels by SP600125. C) MKK7 was not detected in mitochondrial, but in cytosolic fractions. D) The JNK scaffold JIP was present at mitochondria, but 6-OHDA showed no effect on protein levels. Vehicle-treated controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), con (unstimulated control), JIP (JNK interacting protein), JNK (c-Jun N-terminal kinase), MKK4/7 (mitogen activated protein kinase kinase 4/7), SP (SP600125)

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4.4 Inhibition of JNK and ROS production

4.4.1 JNK inhibition does not prevent from oxidative stress

Does the inhibition of JNK also affect ROS levels that are elevated after treatment with

6-OHDA? The JNK inhibitor SP600125, which prevents JNK2 from translocation to

mitochondria, was applied, and DCF fluorescence was measured in the Fluoroskan plate

reader. Higher concentrations of SP inhibitor were used for higher concentrations of 6-OHDA

(2 µM SP in controls, and for 10 µM and 25 µM 6-OHDA, 2.5 µM SP for 50 µM 6-OHDA,

and 3 µM and 4 µM SP for 100 µM and 200 µM 6-OHDA, respectively). Still, SP600125

could not prevent ROS production (Figure 33).

Figure 33. Effect of JNK inhibition on the production of reactive oxygen species

Data was obtained from PC12 cells incubated with H2DCF to assess the amount of reactive oxygen species (ROS) with and without the addition of 6-OHDA for 4 h. ROS production could not be attenuated by the JNK inhibitor SP600125 at any 6-OHDA concentration. Vehicle-treated controls were used. Abbreviations: 6-OHDA (6-hydroxydopamine), FI (fluorescence intensity), H 2DCF (2’,7’-dichloro-dihydrofluorescein), JNK (c-Jun N-terminal kinase), ROS (reactive oxygen species) Statistical significances were calculated using one-way ANOVA with post-hoc Bonferroni. Data are presented as mean ± standard deviation. N = 3.

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4.4.2 Antioxidant mediated neuroprotection against ROS generation

The kavalactone methysticin, the flavon luteolin, and the phytoalexin resveratrol, as well as

tert-butylhydroquinone (tBHQ) were tested for their ability to protect against 6-OHDA- or

MPP+ (1-methyl-4-phenylpyridinium ion)-induced ROS production. 16 h preincubation with

the protectors was followed by 1 h stimulation; 6-OHDA was used in a concentration of

50 µM, MPP+ was applied at 100 µM. Results were correlated to controls. Application of only

the protectors did not change DCF fluorescence compared to controls (data not shown).

Administration of 50 µM 6-OHDA resulted in a 36% ± 8% (P < 0.01), 100 µM MPP+ in a

22% ± 6% increase of ROS levels (P < 0.05).

Higher ROS production following 6-OHDA could be significantly prevented by the

preincubation with all investigated antioxidants (Figure 29A). In detail, 25 µM methysticin

almost completely prevented an increase of ROS (105% ± 8% of control levels; P < 0.01),

preloading with 5 µM luteolin (111% ± 9%; P < 0.05), 5 µM resveratrol (110% ± 6%;

P < 0.05), and 5 µM tBHQ (109% ± 11%; P < 0.05) attenuated ROS elevation effectively as

well (Figure 29A).

MPP+ served as a second model substance to induce oxidative stress in Parkinon’s disease.

MPP+ was administered at a concentration that resulted in a comparable increase of ROS

production as contrasted to 6-OHDA treatment. Statistical analysis revealed that ROS levels

following 100 µM MPP+ or 50 µM 6-OHDA did not differ significantly. Again, methysticin

(97% ± 7%; P < 0.05), luteolin (95% ± 9%%; P < 0.05) and tBHQ (96% ± 9%%; P < 0.05)

prevented ROS generation following the application of the toxin. However, resveratrol was

not effective against MPP+-induced ROS production.

Comparing the generation of ROS in our model, 6-OHDA takes the lead over MPP+ despite

the lower concentration. Antioxidants could effectively prevent the oxidative stress response

to the toxins, with one exception. Resveratrol failed to inhibit ROS production after MPP+.

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Figure 34. Antioxidant mediated protection against reactive oxygen species

The effect of 25 µM methysticin, 5 µM luteolin, 5 µM resveratrol, and 5 µM tert-butylhydroquinone (tBHQ) on ROS levels following stimulation with 6-OHDA (A) or MPP+ (B). Data are presented as the percentage of DCF FI of vehicle-treated controls, as measured in the fluorescence plate reader. Abbreviations: 6-OHDA (6-hydroxydopamine), DCF (2’,7’-dichlorofluorescein), FI (fluorescence intensity), MPP+ (1-methyl-4-phenylpyridinium ion), ROS (reactive oxygen species), tBHQ (tert-butylhydroquinone) Statistical significances were calculated using one-way ANOVA with post-hoc Tukey’s analysis. Data are presented as mean ± standard deviation. N = 4.

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4.5 Involvement of c-Jun N-terminal kinase isoforms in neurite outgrowth

Besides their apoptotic functions, JNKs are essential for the formation and elongation of

neurites (Waetzig and Herdegen, 2003, Gelderblom et al., 2004). To investigate this effect

mouse hippocampi were obtained and separate primary cell cultures were set up and

maintained for up to 6 days. JNK1, JNK2 and JNK3 knock-out (ko) animals and

corresponding wild type (wt) mice were used. To verify the genetic background, mouse

parents and pups were subjected to DNA analysis as described above (materials and methods,

section 3.3.12). Figure 35 shows examplary PCR results (Figure 35).

Figure 35. Genetic background of mice

Exemplary PCR results illustrating the interpretation of mice tissue samples. Double bands correspond to a heterozygous gene pair, whereas a single upper band denotes homozygous wild type JNK, the lower one consists of two truncated gene copies from knock-out mice. Abbreviations: bp (base pairs), JNK (c-Jun N-terminal kinase), WT (wild type)

Wt animals were pooled since they displayed no significant differences between their groups.

One set of Wt samples was exposed to 2 µM of the JNK inhibitor SP600125 immediately

after the explantation. At days 2 and 6 in vitro, cultures were fixed and stained with an

antibody against the neurite marker MAP-2 and neurite lengths were measured. Obtained

values were classified into four groups (lengths of 0 - 39µm, 40 - 79µm, 80 - 119µm, and

>120 µm). The results are brought in Figure 36.

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Figure 36. Effects of JNK isoforms and JNK inhibition on neurite elongation

The effect of total JNK inhibition by SP600125 (SP) and knock-out (ko) of specific JNK isoforms on neurite outgrowth of primary mice hippocampal neurons was analyzed compared to a joint group of their corresponding wild types (WT). Cells were grown for 48 h (A) or 6 d (B), and then stained with the neurite marker MAP-2. Neurite lengths were classified into four groups. SP600125 was applied immediately after the explantation. Abbreviations: JNK (c-Jun N-terminal kinase), ko (knock-out), MAP-2 (microtubule associated protein-2), SP (SP600125), WT (wildtype). * P < 0.05 (compared to corresponding WT group), # P < 0.05 (compared to WT+SP) Statistical significances were calculated using Kruskal-Wallis and Mann-Whithney U test. Data are presented as mean ± standard deviation. N = 3 (minimum).

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After 2 d, wt controls were composed of 65% ± 10% neurites shorter than 40 µm, 29% ± 6%

neurites of 40 - 79 µm length, 6% ± 5% neurites of 80 - 119 µm length, and almost none

longer than 120 µm, yet. Inhibition of total-JNK, as achieved by SP600125, lead to a further

increase within the largest group (<40 µm; 89% ± 11%, P < 0.05), accompanied by the

decline in groups of longer neurites (40 - 89 µm; 9% ± 9%, P < 0.05). In this early stage, none

of the cultures from isoform-specific knock-out mice displayed differences as compared to

controls, only the simultaneous inhibition of all isoforms had an effect (Figure 36A).

When cultivated for 6 d in total, wt hippocampal neurons comprised 33% ± 3% neurites

shorter than 40 µm, 44% ± 4% neurites of 40 - 79 µm length, 16% ± 7% neurites of

80 - 119 µm length, and 8% ± 1% longer than 120 µm. The SP inhibitor substantially

attenuated neurite elongation as demonstrated by less numbers of neurites longer than 80 µm

and 120 µm (7% ± 4%, P < 0.05 and 2% ± 3%, P < 0.05, respectively) and an increase in the

group with shortest neurites (58% ± 3%, P < 0.05). This time, each JNK isoform specific

knock-out alone resulted in a similar pattern as that of total JNK inhibition (as statistical

analysis revealed no differences between these groups). However, regarding the class of

0 - 39 µm neurites, none of the knock-outs could level with the value acquired by SP

incubation (58% ± 3% (SP) versus 49% ± 10% (JNK1), 52% ± 8% (JNK2) or 49% ± 1%

(JNK3)). Additionally, JNK2 and JNK3 ko cells did not show a significant decrease in the

numbers of neurites between 80 µm and 120 µm compared to controls, as it was the case for

total JNK inhibition and elimination of the isoform 1 (Figure 36B).

Taken together, JNKs are needed for neurite outgrowth in vitro. Hippocampal neurons in

culture do not depend on a single JNK isoform for their neurite elongation, although JNK1

shows a slightly greater influence. However, the attenuation of neurite outgrowth by total

JNK inhibition is much more prominent.

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5. DISCUSSION

In the present thesis we sought to investigate the pathological events mediated by the toxin

6-hydroxydopamine (6-OHDA), a model substance to induce PD-like symptoms in vivo and

in vitro. We assessed when and to what extent PC12 cells die following the administration of

6-OHDA. We analyzed the redox status over 24 h, measuring reactive oxygen species (ROS)

by rather compartment-specific fluorescent dyes, and checked the integrity of the

mitochondrial membrane potential (∆ΨM). Mitochondria are the most relevant sites of ROS

generation, especially when ∆ΨM is disrupted. The loss of mitochondrial integrity leads to

release of certain pro-apoptotic proteins into the cytosol, and cell death signaling via caspases

reaches a point of no return. We therefore also measured the levels of cytochrome c, which is

an important constituent of the mitochondrial respiratory chain, but induces cell death when in

contact with the cytolsol by forming the apoptosis protease activating factor-1 (APAF-1).

Filling the gap between oxidative stress and mitochondria-mediated cellular death, we

investigated the role of c-Jun N-terminal stress kinases (JNK). There are numerous reports

addressing a role to JNK-induced release of pro-apoptotic factors from mitochondria (see

below). Thus, we looked at the isoform-specific expression of JNK in isolated PC12 cell

mitochondria, but also in the nucleus where JNK is supposed activate a variety of

transcription-factors, including AP-1 and c-Jun . Results from this thesis demonstrate a role of

JNK isoform 2 (JNK2) in the vicinity of mitochondria. The inhibition of JNK protected PC12

from events following downstream of/after JNK activation, however, it did not exert any

effect on 6-OHDA-induced ROS. Instead, four antioxidants (methysticin, luteolin, resveratrol

and tert-butylhydroquinone (tBHQ).

5.1 6-OHDA-induced cell death

Previous studies showed that 6-OHDA induces apoptosis in various model organisms. Among

these were hemiparkinsonian rats, with injection to the median forebrain (He et al., 2000), the

striatum (Mladenovic et al., 2004), and the substantia nigra (Jeon et al., 1999), as well as

different cell types, including the rat pheocromocytoma PC12 cells (Walkinshaw and Waters,

1994; Blum et al., 1997; Ochu et al., 1998; Takai et al., 1998; Hanrott et al., 2006), the

human neuroblastoma SH-SY5Y cell line (von Coelln et al., 2001; Jordan et al., 2004), the

dopaminergic (DA) cell line MN9D, a fusion of ventral mesencephalic and neuroblastoma

cells of human origin (Choi et al., 1999; Oh et al., 1998), and primary DA neurons from rats

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and mice (Han et al., 2003; Lotharius et al., 1999). DNA fragmentation and chromatin

condensation, caspase activation, p53- and bax expression, PARP cleavage and the release of

cytochrome c from mitochondria were taken as evidence for apoptotic processes in these

models. In contrast to the low molar range (10-50 µM), doses of 100 µM 6-OHDA and more

result in distinct features of necrotic cell death, e.g. loss of membrane integrity (Ochu et al.,

1998). The discrete signaling cascades of the apoptotic machinery remain the targets for

pharmacons to stop PD in patients.

In the present thesis, 6-OHDA concentrations ranged from 10 µM to 200 µM. PC12 cell death

was assessed by fluorescence of the apoptotic marker Annexin V and propidium iodide (PI), a

marker for necrosis, in the flow cytometer. Following our protocol (see section 3.3.7.4), it was

not possible to point out a specific dividing rule at what concentration necrosis, as measured

by PI, would occur. In all 6-OHDA samples a small proportion of PI fluorescent events,

though with an increasing tendency towards higher concentrations, was detectable (data not

shown).

We propose that a loss of membrane integrity, which is necessary for PI to be detected in the

flow cytometer, is an integral part of 6-OHDA mediated toxic events. Only few studies have

analyzed direct effects of 6-OHDA to the cell membrane. Berretta et al. found that 6-OHDA

rapidly damages the cell membrane, with special regard to distal dendrites of SNpc neurons.

Blockade of the dopamine transporter (DAT) did not prevent membrane disturbances

(Berretta et al., 2005). However, the specificity of 6-OHDA towards dopaminergic cells via

the uptake by the DAT is controversial. Due to the similarity to dopamine there is no doubt

that 6-OHDA can be taken up by the DAT. On the other hand, there are several studies

pointing out that 6-OHDA toxicity cannot be prevented by inhibition of the uptake (Abad et

al., 1995; Blum et al., 2000; Hanrott et al., 2006), and that 6-OHDA mediated cell death is

not restricted to DA neurons (Blum et al., 2000; Dodel et al., 1999; Lotharius et al., 1999;

Seitz et al., 2000). Additionally, the administration of catalase, an enzyme of molecular

weight so large that it cannot enter the cell under normal circumstances, strongly protects

against the toxin (Abad et al., 1995; Yamada et al., 1997; Hanrott et al., 2006).

In our cell model, incubation with 50 µM 6-OHDA leads to a significant loss of viability after

24 h (72% ± 6% of vehicle-treated controls). Comparisons with other published data of

6-OHDA toxicity in cell models can only be drawn with caution as to the concentration and

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the cell loss. Many studies analyze other apoptotic markers than Annexin V/PI (e.g. tyrosine

hydroxylase↓, TUNEL, MTT assay, trypan blue staining, etc.), and different apoptotic

markers give different results (Kylarová et al., 2002). 6-OHDA is an agent that is susceptible

to autoxidation, so it is very important to know how it was applied and stored. Unfortunately,

this information is often unsatisfactory. Additionally, many researchers tend to use 100 µM

6-OHDA or even more. The effect of the toxin depends also very much on the cell type due to

their different origins, culture conditions and expression patterns. There are even differences

between younger and older cells in culture from the same cell line, and between controls and

cells transfected with a vehicle. However, 6-OHDA-induced cell death has been analyzed by

Annexin V/PI staining in the flow cytometer by three research groups, Salinas et al. found

12% Annexin V-positive PC12 cells after 6 h of 40 µM 6-OHDA (Salinas et al., 2003), Ebert

et al. analyzed MN9D cells and saw 35% apoptotic neurons four hours after 100 µM 6-

OHDA (Ebert et al., 2005), and Guo et al. made a very thorough time-line and concentration

analysis of 6-OHDA in SH-SY5Y cells, with 81% ± 8% surviving following 50 µM 6-OHDA

for 24 h (Guo et al., 2005). The results of time- and concentration-dependency of cell death

induced by 6-OHDA in this thesis are generally in line with results obtained by Guo et al.,

despite the fact that they investigated another cell line. Other data from PC12 cells (mainly

MTT viability assays) that show 6-OHDA toxicity in a concentration-dependent and time-

resolved manner are congruent with our findings (Hanrott et al., 2006; Saito et al., 2007).

5.2 6-OHDA toxicity to mitochondria

From the variety of apoptotic pathways, our data provides evidence that mitochondria are a

target of 6-OHDA toxicity. The release of mitochondrial cytochrome c into the cytosol is the

key event of the intrinsic pathway (Liu et al., 1996; Kantrow and Piantadosi, 1997; Kluck et

al., 1997). Thereafter, this has been named the “point of no return” in the apoptotic process,

since cytochrome c is a constituent of the apoptosome that activates caspases (Li et al., 1997;

Zou et al., 1999; Kroemer and Reed, 2000). However, cyt c is abundant in the mitochondrial

intermembrane space (Forman and Azzi, 1997), and there is no evidence, that the pool of

cyt c, which resides in the cristae as a participant in the respiratory chain, is released in early

apoptosis (Waterhouse et al., 2001). It seems that this pool requires more drastic remodelling

of the mitochondrial structure, which eventually occurs in the course of mitochondrial

damage, leading to the loss of ∆ΨM (Scorrano et al., 2002). We investigated the mitochondrial

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membrane potential in our model, and found that 6-OHDA induced a clear decline after 8 h,

which is coinciding with the release of cytochrome c into the cytosol (25 µM). Low

concentrations of 6-OHDA (10 µM) did not have an effect, ∆ΨM dropped at the 8 hour time

point but then was stabilized following medium doses (25 µM, 50 µM), whereas high level

6-OHDA incubation led to a consecutive decline over the investigation period. These results

can be explained by studies that describe the mitochondria as a checkpoint, that is able to

compensate and intensify death signals, dependent on the strength and duration of the insult

(Nicholls and Budd, 2000).

In isolated mice brain mitochondria 6-OHDA even promotes a faster and more profound

disruption of ∆ΨM. Possible reasons for these findings could be that cytosolic 6-OHDA- and

ROS-detoxifying enzymes are missing in the mitochondrial fractions, or that 6-OHDA/ROS

exert direct membrane-disturbing activities which now fully target to mitochondrial

membranes alone, and not to the cell membrane as proposed by early PI accumulation (see

above) or to vesicular membranes. Not only is the cytosol equipped with strong antioxidant

enzymes and radical scavengers compared to the mitochondrial matrix or the intermembrane

space, but mitochondria are naturally more exposed to oxygen radicals. Particularly isolated

mitochondria in a non-optimal environment are more vulnerable. However, the results

underline the strong toxicity of 6-OHDA to mitochondria.

In experiments to analyze its effects on ∆ΨM in a continuous manner, 6-OHDA was

administered to PC12 cells and isolated mice brain mitochondria. Surprisingly, we observed

an immediate ∆ΨM lowering effect. Generally, 6-OHDA has been shown to impair ∆ΨM in

preparations of isolated rat brain mitochondria (Lee et al., 2002). In that study, safranin O was

used as a, however, there are no studies that used the dye JC-1 in the fluorometer to analyze

isolated mice brain mitochondria after 6-OHDA incubation. Our results from isolated

mitochondria are not in contrast to studies using the flow cytometer, since the time points are

not overlapping. Furthermore, in the experiments with isolated mitochondria in the

spectrofluorometer, complex I is passed by inhibition by rotenone and providing complex II

with the natural substrate succinate. So one could speculate that this immediate disruption is

due to an inhibition of complex II, III or IV of the respiratory chain, an oxidative attack to the

mitochondrial membrane followed by ROS scavenging, or a direct interaction of 6-OHDA

with the red fluorescent J-aggregates in the mitochondrial matrix, resulting in the transient

dissociation to green monomers. However, the acidic properties of 6-OHDA are in contrast to

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this theory, because J-aggregates form in the presence of protons (Cossariza et al., 1996). We

hypothesize, that an intracellularly produced, unknown metablite or peroxide from

autoxidation/metabolism processes targets complex I, or 6-OHDA/ROS oxidize essential

proteins of the mitochondrial membrane leading to the decrease of ∆ΨM.

5.3 Profound and early oxidative stress in PC12 cells, but not isolated

mitochondria following 6-OHDA

Oxidative stress has been documented throughout the years of research in PD (Götz et al.,

1990; Jenner, 2003). From the model toxins investigated, 6-OHDA exerts the highest impact

of oxidative stress, whereas other toxins that target the respiratory chain (e.g. MPP+) show

less oxidative burden (Choi et al., 1999). In agreement with this, our results show that MPP+

could only increase ROS levels when administered in a concentration twice as high as

compared with 6-OHDA. The latter induces ROS production in virtually all models, in vivo

(Perumal et al., 1989; Kumar et al., 1995) as well as in vitro (Decker et al., 1993; Abad et al.,

1995; Choi et al., 1999; Lotharius et al., 1999; Hanrott et al., 2006). Also, the recent

descriptions of a genetic predisposition leading to higher susceptibility of PD patients towards

oxidative stress has strengthened the importance of ROS in the development of the disease

(Zimprich et al., 2004).

ROS production induced by 6-OHDA originates from the following three main sources: (I)

ROS generation by autoxidation, (II) H2O2 generation after deamination by monoamine

oxidase (MAO), and/or (III) direct inhibition of the mitochondrial respiratory chain (for

review see Blum et al., 2001a).

This study demonstrates that ROS cannot be generated by 6-OHDA concentrations lower than

200 µM in isolated mice brain mitochondria. The uncoupling properties of 6-OHDA cannot

play a role here because they have been described to come into play primarily in very high

concentrations of 6-OHDA (Brand, 2000; Thakar and Hassan, 1988). However, the relevance

of ROS generation following 6-OHDA is not of question (see above). Our results show that in

whole PC12 cells oxidative stress is an early and dominant mediator of 6-OHDA toxicity. A

lot of studies have used high concentrations of 6-OHDA in their systems (e.g. 100 - 200 µM).

Here we show that doses of 50 µM, 100 µM and 200 µM are comparable after an incubation

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of 4 h regarding the elevation of ROS levels. The observation of ROS production over time

revealed a great increase within the first 2 h, which was followed by a second boost between 8

and 16 h of treatment. A very much similar pattern has been described by Choi et al. in

MN9D cells using 100 µM 6-OHDA (Choi et al., 1999).

The major intracellular source of ROS are mitochondria. We were interested where the first

ROS are generated in our model. In comparison between mitochondrial and cytosolic ROS-

sensitive dyes our data point to a major contribution of mitochondrially originated ROS

during the early boost. The specificity of ROS-sensitive dyes, however, is still a matter of

debate, so the argument of mitochondrial ROS generation might require additional proof. In

fact, a number of studies have demonstrated that 6-OHDA does not induce toxicity either by

direct mitochondrial inhibition or by enzymatic deamination by MAO, but via an extracellular

mechanism (Blum et al., 2000; Hanrott et al., 2006; Soto-Otero et al., 2000). Inhibitors of

dopamine and noradrenaline transporters failed to prevent PC12 cells from ROS generation,

cytochrome c release and cell death (Hanrott et al., 2006; Saito et al., 2007).

But also extracellular H2O2 generation by autoxidation alone cannot explain the mechanism of

6-OHDA toxicity, since hydrogen peroxide degradation by catalase does not result in full

protection (Saito et al., 2007). When adding 6-OHDA to our preparations of isolated

mitochondria, the autoxidation process, which is still likely to occur, does not induce ROS

from mitochondria. Among the autoxidation products are H2O2 and cyclization products

(semi-quinones, quinones, aminochromes, melanin). From all known dopamine analogues

6-OHDA is most susceptible to autoxidation (Izumi et al., 2005). Autoxidation of 100 µM

6-OHDA occurs rapidly, the toxin is completely oxidized within 3.5 h when given to PC12

cells (Blum et al., 2000). In the human disease, however, 6-OHDA is a very rare metabolite of

dopamine, addressing a high impact of oxidative stress to dopamine autoxidation itself.

Nevertheless, autoxidation products of dopamine and also 6-OHDA play a major role in PD

cell models (Arriagada et al., 2004; Asanuma et al., 2004). High toxin concentrations

administered to isolated mitochondria do result in ROS production also in our system. We

hypothesize that sufficiently high concentrations can induce ROS generation, low level

6-OHDA incubation may be intensified by cell membrane or cytosolic compounds, which are

lost in mitochondrial homogenates. Dopamine (DA) and DA analogues-toxifying enzymes

COX-2 (Tyurina et al., 2006), tyrosine hydroxylase (Arriagada et al., 2004) and cytochrome

P450 (Bernhardt et al., 1996) have been addressed a role in PD pathology. To date, the

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116

intermediate products p-quinone and the highly reactive leukoaminochrome radical from the

metabolic pathway target cellular vital functions, and pose a high oxidative threat to essential

proteins (Watanabe and Forman, 2003). A facilitated conversion to the end-product melanin

could thereby provide some protection, though melanin complexes iron that in turn can

increase the oxidative burden (Izumi et al., 2005).

Our findings lead to the conclusion that (I) 6-OHDA toxicity cannot only be dependent on

autoxidation, as suggested also by Saito et al. (Saito et al., 2007), (II) MAO activity in the

outer mitochondrial membrane may only be relevant in ROS production following higher

6-OHDA concentrations (Hanrott et al., 2006; Soto-Otero et al., 2000), and (III) there is also

no evidence for a direct effect of 6-OHDA on the respiratory chain in concentrations less than

200 µM (conflicting data on the relevance of complex I inhibition from Glinka et al., 1996

and Storch et al., 2000). An explanation could be that a metabolite of 6-OHDA is responsible

for targeting complex I, that would be generated intracellularly and not extracellularly. During

our studies with the toxin, we have seen that PC12 cells do not undergo apoptosis if 6-OHDA

is administered in its oxidized form (data not shown; see also Pedrosa et al., 2002), and

reports point out, that H2O2 alone does not fully represent 6-OHDA-induced pathological

events (Saito et al., 2007).

5.4 6-OHDA-induced JNK2 activation and translocation to mitochondria

Toxic features of 6-OHDA involve the activation of c-Jun N-terminal kinases in a variety of

cell types, including the dopaminergic MN9D cell line (Choi et al., 1999), and SHSY-5Y

cells (Ha et al., 2003). The inhibition of JNK is protective against caspase activation and

apoptosis induced by cytotoxic agents in Jurkat cells (Krilleke et al., 2003), rat intestinal

(Bhattacharya et al., 2003) and human colon cancer cells (Xiao and Liu, 2003). Furthermore,

JNK are strong activators of mitochondrial stress and the release of mitochondrial

apoptogenic proteins (Bhattacharya et al., 2003; Blum et al., 2001a; Krilleke et al., 2003;

Okuno et al., 2004; Tournier et al., 2000; for review see Waetzig and Herdegen, 2005). The

relevance of JNK inhibition for PD has been demonstrated for various in vitro and in vivo

models of the disease (for reviews see Wang et al., 2004; Kuan and Burke, 2005; Borsello

and Forloni, 2007). However, inhibitors like SP600125 that target the common activation site

of all JNK isoforms have failed in early clinical studies (Waldmeier et al., 2006).

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Nonetheless, isoform-specific inhibition, small peptide drugs that bind outside the ATP-

docking site, or upstream kinase inhibition may still provide breakthrough potential in

pharmacological intervention of apoptotic processes (Barr et al., 2002; Kuan and Burke,

2005).

6-OHDA-induced activation of JNK in PC12 mitochondria was first described by our group

(Eminel et al., 2004). PC12 cells are a cell line of peripheral origin, so only JNK1 and 2 are

expressed (Butterfield et al., 1999; Mielke et al., 2000). Since the JNK isoform specific

antibodies on the market allow only for differentiation between JNK1 and JNK2/3 (Coffey et

al., 2002), this model allows us to detect specific changes of JNK1 and 2. Several studies

have proposed that JNK effects on mitochondria would need JNK in close vicinity to this

organelle, before Kharbanda et al. actually found JNK present and increasing in

mitochondrial fractions following ionizing radiation (Kharbanda et al., 2000). We were

interested if JNK would also be detectable in isolated mitochondria from PC12 cells, if this

pool of JNK was reflected by a specific isoform, and if JNK isoform levels reacted

substantially and specifically to 6-OHDA treatment.

Under basal conditions, JNK are found in mitochondrial preparations. Our results attribute

this pool to consist mainly of JNK1. Mitochondria-associated JNK was strongly activated in

response to 6-OHDA within 4 h and continued to be active up to 24 h. This activation was

accompanied by a translocation of JNK2 into the nucleus and to the mitochondria. Since the

expression of phosphorylated JNK cannot be determined in an isoform-specific way, we

conclude by the timely overlap of simultaneous activation and JNK2 increase in

mitochondrial fractions, that JNK2 is the isoform that is mainly phosphorylated and is

responsible for further signal transduction in this organelle. Transfection of PC12 cells with a

dominant-negative mutant JNK2 (dnJNK2) produced an altered gene product, that acted

antagonistically to the wild-type allele, and reduced the pool of phosphorylated JNK in the

nucleus after stimulation with 6-OHDA and strongly reduced the 6-OHDA-induced

translocation of endogenous JNK2 to the nucleus while dnJNK1 had not such effect (data

from our group, Eminel et al., 2004). Similarly, JNK inhibitor SP600125 attenuated the

translocation of JNK2, but not JNK1 into the nucleus following 6-OHDA treatment (Eminel

et al., 2004). The translocation of JNK2 to the mitochondria supports the notion that the

assembly of a selective JNK2 signalosome propagates the mitochondrial pathology. A central

role of JNK2, but not JNK1, for cellular degeneration was seen in fibroblasts where JNK2

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mediates tumor necrosis factor-alpha-induced cell death (Dietrich et al., 2004). Similarly,

JNK2/3 have been addressed an impact for neuronal stress in primary cerebellar granule

neurons (Coffee et al., 2002). Recently, it was shown that in various cell types including

fibroblasts, erythroblasts and hepotocytes, JNK2 deficiency leads to increased cellular

proliferation (Sabapathy and Wagner, 2004). Importantly, SP600125, which prevents the

stress-induced alterations in the membrane potential (Sanna et al., 2002), blocks the

mitochondrial translocation of JNK2, but not JNK1. This finding suggests that intracellular

distribution of JNK2 depends on its activation and, by a positive feedback mechanism, on

activated upstream kinases (Holtz et al., 2003). So altogether, our findings suggest that JNK2

is the active JNK isoform which mediates the neurodegenerative effects of 6-OHDA in vitro.

5.5 Intracellular JNK pools

The presence of JNK1 did not change in mitochondria, but was amplified in cytosol and

nucleus upon the administration of 50 µM 6-OHDA. JNK2 levels remained low in cytosol

and nucleus. Cell stress signaling, e.g. by JNK, results in death and differentiation (as

discussed below). Therefore, specific actions should be mediated by individual isoforms in

specific parts of the cell. The intracellular localization may be the ultimately critical factor.

For example, JNK1 is an important mediator of insulin resistance associated with obesity

(Hirosumi et al., 2002; Kaneto et al., 2004), but it is also indispensable for the intact

cytoarchitecture of the brain (Chang et al., 2003). JNK2 is recruited by apoptotic stimuli

(Coffee et al., 2002; Dietrich et al., 2004), but it is also important for the coordinated

differentiation of, for example, activated immune cells (Jaeschke et al., 2004), and for brain

development (Kuan et al., 1999). JNK3, in particular, is considered to be a potent effector of

neuronal death (Yang et al., 1997; Hunot et al., 2004; Brecht et al., 2005). However, as proof-

of-principle of the context-specific functions of JNK variants, a splice variant of JNK3

mediates neurite outgrowth in addition to stress-induced apoptosis in PC12 cells (Waetzig and

Herdegen, 2003).

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5.6 Upstream and downstream effectors of JNK signaling at the

mitochondria

The specific mitochondrial translocation of JNK2, but not JNK1, raises the question which

signaling pathways control the JNK translocation to and/or the JNK activation at the

mitochondria. In leukaemia cells, the phosphorylation of JNK is a prerequisite for trans-

location which is supported by the absence of JNK2 in the mitochondria (Ito et al., 2001). In

our model, MKK4 and JIP-1 are localized at the mitochondria, whereas MKK7 was not

detectable in the mitochondrial fraction. This points to a specific JNK pathway in

mitochondrial pathology. MKK4 was previously found in mitochondrial fractions from

cardiomyocytes (Okuno et al., 2004), whereas the presence of JIP-1 and the absence of

MKK7 are novel observations. The absence of MKK7 in mitochondrial preparations is also

comprehensible when considering findings, that the MKK7/JNK pathway is responsible for

neuritogenesis and neurite regrowth after injury (Hidding et al., 2005; Coffee et al., 2000).

The scaffold protein JIP-1 forms complexes with JNKs and selected members of the upstream

phosphorylating kinases (Dickens et al., 1997; Whitmarsh et al., 1998). It is not a counter-

argument against a translocation of the MKK4-JNK2-JIP complex to mitochondria that JIP

levels do not obviously change in mitochondrial fractions, but are kept in balance. In fact,

JIPs have been found to direct JNK and their upstream kinases to different compartments of

the cell by associating with microtubules (Goldstein, 2001; Verhey et al., 2001). We believe

that JNK2 activation in the cytosol, and/or effects on anchor proteins would target the

signalosome to the mitochondria.

Our data sets the release of cytochrome c downstream of JNK activation and translocation of

JNK2 to mitochondria, since this event can be inhibited by SP600125. What may be the

connection between JNK and cytochrome c release? The proapoptotic Bax resides in the

cytosol due to the binding to an isoform of the 14-3-3 anchor protein (Nomura et al., 2003).

Upon phosphorylation of this cytosolic anchor by phospho-JNK the complex dissociates and

Bax translocates to the mitochondria where it forms channels that would allow cytochrome c

to leave the intermembrane space and establish the apoptosome leading to cell death (Tsuruta

et al., 2004; Nomura et al., 2003; Putcha et al., 1999). The JNK inhibitor SP600125 prevented

bax translocation to mitochondria (Tsuruta et al., 2004). Before that study, there were already

publications showing that JNK regulates Bax translocation via the phosphorylation of Bim

after trophic factor withdrawal (Lei and Davis, 2003; Putcha et al., 2003). In contrast to these

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120

results, Tsuruta et al. saw no effect of either SP600125 or the overexpression of a

constitutively active JNK on the activation of Bim (Tsuruta et al., 2004). Following 6-OHDA

our group also saw no increase in bim mRNA levels, but the inhibition of JNK by dnJNK2

reduced bim expression (Eminel et al., 2004). These results can not ultimately determine the

role of Bim in 6-OHDA-induced, JNK-mediated cell death. However, bax is strongly

activated by the administration of 6-OHDA to PC12 cells (Blum et al., 1997), and bax pores

could lead to the release of cytochrome c, that we found subsequently to JNK activation and

translocation.

JNK activity also targets the group of anti-apoptotic bcl-2 family members, Bcl-2 and Bcl-XL.

These proteins locate in mitochondrial, nuclear and ER membranes (Blagosklonny, 2001).

Their main function is to prevent the assembly of bax proteins and the formation of a pore,

thereby protecting mitochondria from mitochondrial membrane disruption and release of

apoptogenic factors (Gross et al., 1998; Nomura et al., 1999; Yang et al., 1995). JNK

phosphorylation of Bcl-2 and Bcl-XL leads to a decrease in the levels of Bax heterodimers

with Bcl-2 or Bcl-XL, and promotes the formation of potentially toxic bax homodimers, which

form bax pores channeling e.g. cytochrome c into the cytosol (Kharbanda et al., 2000). Cyt c

levels became elevated following 6-OHDA treatment in our model way after the activation of

JNK and translocation of JNK2 to mitochondria. So rather the inhibition of mitochondrially

residing anti-apoptotic Bcl-2 and Bcl-XL than the cytosolic activation of Bax serves as an

explanation of JNK2 apoptotic functions in mitochondrial preparations of PC12 cells.

What happens upstream of JNK2 activation and translocation to the mitochondria? Our data

provides evidence that from the two upstream kinases of JNK (Ip and Davis, 1998) only

MKK4 is involved. The activation of MKK4 is controlled by MEKK1/4, the mixed-lineage

kinases (MLK) 2/3 and apoptosis signal-regulating kinase 1 (ASK1) (for review see

Schlesinger et al., 1998; Chang and Karin, 2003). Very recently, Chen et al. described the

protective effects of adenoviral transfer of dominant-negative constructs from the dual-leucine

zipper kinase (DLK) of the MEKK family after 6-OHDA lesions in mice (Chen et al., 2008).

The role of MLK3 has been investigated in mice and rats treated with 6-OHDA, however,

results are conflicting (Chen et al., 2008; Pan et al., 2007). ASK1 is a strong regulator of

MKKs and JNKs. Ouyang et al. found that inhibition of ASK1 activity by siRNA or by

overexpression of a kinase-dead mutant protected SHSY-5Y cells from 2 h incubation of

100µM 6-OHDA (Ouyang et al., 2006). The authors also showed that ROS scavenging

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121

reduces phosphorylation of ASK1. This sheds new light on the mechanism of JNK activation

by ROS (Crossthwaite et al., 2002; Ouyang et al., 2006).

5.7 Time course of pathological events mediated by 6-OHDA

Our studies aimed at giving an overview of events in one model, that occur due to the

treatment with 6-OHDA and may shed more light on the mechanism by which the toxin

mediates these actions. We used mainly 25 µM or 50 µM 6-OHDA, which is lower than the

concentration used in most other studies, while investigating the following parameters: cell

death, ROS production, disturbances of the mitochondrial membrane potential, cytochrome c

release, JNK activation and localization.

Figure 37. Time course of events in 6-OHDA mediated cell death

Time course depicting the parameters analyzed in this study following the administration of 6-OHDA. ROS production is increased first, then the upstream kinase MKK4 appears activated at mitochondria, followed by phosphorylated JNK, and an increase of JNK2 in mitochondrial preparations. Sub-sequently, the mitochondrial membrane potential is disrupted, and cytochrome c is released. Cell death occurs at around 24 h, after ROS have peaked. The * denotes the first significant change compared to vehicle-treated controls. Abbreviations: ∆ΨM (mitochondrial membrane potential), JNK (c-Jun N-terminal kinase), MKK4 (mitogen-activated protein kinase kinase 4), ROS (reactive oxygen species).

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Our results demonstrate a profound and early increase of reactive oxygen species (60 min),

ROS levels stabilize between 4 and 8 hours, then a second significant increase can be

observed over a period of 8 hours (Figure 37).

The source for ROS generated within 60 min appears to be mitochondrial. So the

mitochondrial pathology is already ongoing, when the upstream kinase MKK4 and JNK are

activated (within 4 hours), and levels of the JNK isoform 2 increase in mitochondrial fractions

by the same time. We can only speculate here, how ROS activate MKK4 and JNKs, e.g. via

ASK1 (Ouyang et al., 2006). The activation, translocation to mitochondria and the pore

formation of bax, mediated by JNK and ROS (Okuno et al., 2004; Putcha et al., 2003; Tsuruta

et al., 2004), serves as an explanation to the subsequent release of cytochrome c (supposingly

through bax pores). This is accompanied by a disruption of the mitochondrial membrane

potential when incubated with 6-OHDA for 8 hours. At the 16 hour time point ROS peak; in

the following 8 hours PC12 cells start to die, by about 30% (data from 50 µM 6-OHDA

treatment).

5.8 JNK inhibition and protection from oxidative stress

JNK inhibition is protective against PC12 neuronal death (Fujita et al., 2006; Ito et al., 2006;

Marques et al., 2003) and is regarded as a therapeutic strategy in PD (Kuan and Burke, 2005),

as well as against the release of cytochrome c from mitochondria, as shown by us and other

groups (Eminel et al., 2004; Guan et al., 2006; Krilleke et al., 2003). Furthermore, we could

demonstrate that SP600125 inhibited also the activation of the JNK upstream kinase MKK4

plus the translocation of JNK2 to mitochondria. However, JNK inhibition did at no

concentration prevent from 6-OHDA-induced ROS elevation in 4 hours. Since SP600125

does not exert any antioxidant features itself, this shows distinctly that ROS are upstream of

JNK activation and translocation to mitochondria. The JNK inhibitor is not able to target JNK

isoform-specifically, so we need to accept that the protective effects could be counteracted by

suppression of physiological features of JNK, as JNK inhibition also dramatically impairs

neurite outgrowth of hippocampal neurons (as discussed below).

Therefore, we tested the kavalactone methysticin, the flavon luteolin and the phytoalexin

resveratrol, as well as tert-butylhydroquinone (tBHQ), which has been already shown to

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protect SHSY-5Y cells against 6-OHDA-induced ROS production, JNK activation and cell

death (Hara et al., 2003). Additionally to radical scavenging properties the authors described

the activation of the antioxidant response element (ARE), that regulates the expression of

enzymes relevant in the cellular defense system. Just recently, our group also discovered that

luteolin activates protective genes via the Nrf-2-ARE pathway (Wruck et al., 2007). The

results of this thesis show that preincubation with all four protectors prevented the rise of

ROS induced by 50 µM 6-OHDA. Even though the antioxidants were not present anymore

when the toxin was given (due to washing steps) and the samples were analyzed, they were

protective against oxidative stress. We propose a similar lasting effect on cellular defense

mechanisms, provided by the preincubation with these compounds, as our group could

already demonstrate for luteolin (Wruck et al., 2007). In comparison, incubation of PC12

cells with methysticin, luteolin and tBHQ protects from ROS generation induced by the

neurotoxicant MPP+ (100 µM). Resveratrol does not confer any protection from MPP+-

induced ROS. However, despite using double the concentration the increase in ROS

production after MPP+ is little (ca. 20% vs. 40% of 6-OHDA). It has been noted before that

oxidative stress is a less relevant part of the toxin’s mechanism to induce dopaminergic cell

death (Choi et al., 1999; Fall et al., 1999; Wadia et al., 1998). Following MPP+ and the

precincubation with all protectors, their ROS levels did not differ significantly from controls.

We conclude that the precincubation with methysticin, luteolin, resveratrol and tBHQ confers

increased cellular protection against to oxidative stress induced by 6-OHDA. Furthermore,

our results, based on our experimental setup, suggest that it is not only a scavenging effect,

but possibly also other pathways such as the activation of the transcription factor Nrf-2 and

consequent upregulation of cytoprotective gene products (e.g. glutathion synthetase).

5.9 JNK in neuronal death and survival

Therapeutic JNK inhibition may come along with side effects, since up to date, also

physiological functions of JNK can not clearly be distinguished from pathological features

(for review see Waetzig and Herdegen, 2005). Our investigations addressed the question to

which extent JNK stresskinases are involved also in neurite outgrowth, the main

morphological feature of neuronal differentiation and repair in the rodent brain.

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We dissected murine neonatal brains, obtained and cultivated hippocampal neurons, which

express all JNK isoforms including JNK3. Inhibition of JNK activity by SP600125

dramatically attenuates the formation of longer neurites after 48 h and 6 d in vitro. Primary

cell cultures from JNK1 ko, JNK2 ko and JNK3 ko mice revealed that all three JNK isoforms

contribute to neurite outgrowth. The JNK1 ko provided the most pronounced impairment and

this corresponds to its role during development and for maintenance of the neuronal

cytoskeleton (Chang et al. 2003; Björkblom et al. 2005). Our group has shown in PC12 cells

(which are devoid of JNK3) that JNK2, but not JNK1, triggers neurite regrowth following

injury (Waetzig and Herdegen, 2005). The analysis of the role of JNK3 for regrowth was

complicated by the finding that the absence of JNK3 handicapped the success of keeping

these cells in culture. The question remains whether JNK3 deficiency only affects neurite

outgrowth and/or primary cell growth and adhesion. In any case, the sensitivity of

hippocampal neurons for JNK3 deficiency correlates with the findings that a truncated JNK3

mutant with loss of the activation domain is not compatible with the survival of PC12 cells;

importantly, this truncated JNK3 mutation provokes severe neurological symptoms in humans

(Shoichet et al. 2006). These observations confirm that besides JNK3, JNK2 is involved in

physiological and regenerative responses. Total JNK inhibition by SP600125 leaves only

about 10% of longer neurites (> 40 µm) after 2 days, which is in contrast to controls and

knock-out of an individual isoform at that stage (ca. 40% neurites > 40 µm). After 6 d in

culture, JNK isoforms do not seem to be able to compensate for the lack of one of them

anymore. All knock-outs have shorter neurite length as compared with controls. The results

of this study account for a profound involvement of JNK in neuronal sprouting with no

regards to a specific isoform.

Our microscopical analyses reveal a substantially large pool of JNK1 in the nucleus of control

PC12 cells. The intensity of the FITC-marked isoform increases slightly upon stimulation

with 6-OHDA, and there was also an increase of JNK2 in the nucleus. The activity of JNK in

the nucleus is connected with the pro-apoptotic actions of JNK (Björkblom et al., 2005), and

inhibition of JNK activity by SP600125 was reported to increase neurite outgrowth in

embryonic cerebellar neurons (Coffey et al. 2000). However, JNK activity is the major trigger

of neurite outgrowth and regrowth even dominating the role of ERK (Kuan et al., 1999;

Sabapathy et al., 1999). And the transcription factor c-Jun, wich is mainly but not only

activated by JNK in the nucleus, is imperative for the regeneration of axons in vivo (Raivich

DISCUSSION

125

et al. 2004) and can be linked to outgrowth of axons, dendrites and neurites (Besirli et al.

2005; Herdegen et al. 1998; Lindwall and Kanje, 2005).

How can JNK signaling selectively activate either neuronal (apoptotic death) or

regrowth/regeneration? This dichotomy can not be attributed to individual JNK isoforms, but

might be inherent in one JNK isoform (for review see Waetzig and Herdegen, 2005). We

conclude that in the highly plastic hippocampal neurons, all JNKs participate in neurito-

genesis. In consequence, the search for the side-effect free inhibitor of ‘the apoptotic’ JNK

isoform appears yet to be an illusion which requests different therapeutic strategies against the

neurodegenerative impact of JNK (Waetzig and Herdegen, 2005).

5.10 Technical considerations

5.10.1 The PC12 cell model

The rat pheochromocytoma cell line PC12 is widely used to study neurodegeneration and

neuronal differentiation in vitro (Shafer et al., 1991). Upon stimulation with nerve growth

factor (NGF) PC12 cells elongate their processes and display a neuronal phenotype (Greene

and Tischler, 1976). In this study we used undifferentiated PC12 cells, because their

enzymatic composition and channel expression has been found to be higher in regard to

dopamine metabolism (Shafer et al., 1991), more reliable (Woodgate et al., 1999) and

experiments with these cells more reproducible. Of note, undifferentiated PC12 cells are more

susceptible to toxic stimuli, while NGF is protective to the cells (Salinas et al., 2003;

Woodgate et al., 1999).

5.10.2 Preparation of isolated mitochondria

The protocols for the preparation of isolated mitochondria from PC12 cells and mice brains

were optimized independently to minimize cytosolic contaminations. Usually, ß-actin was

present in low-level concentrations even in highly purified mitochondria. This can be

explained by the tight connections between mitochondria and the cytoskeleton together with

the nature of the isolation method. We consider low-level contamination of mitochondrial

preparations with the abundant ß-actin inevitable against the background of possible

functional damage to the organelle, if the isolation procedure increases with time. However,

expression levels of JNK found in mitochondria are higher than could be explained by

DISCUSSION

126

cytosolic origin, and higher than comparable ß-actin expression from the same sample, so that

we are certain, that JNK either anchores in the outer mitochondrial membrane, or enters the

intermembrane space.

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ABBREVIATIONS

158

7. ABBREVIATIONS

∆ΨM delta psi, mitochondrial membrane potential

6-OHDA 6-hydroxydopamine

ABC avidin-biotin-complex

AP-1 activator protein-1

APS ammonium persulphate

AraC Cytosine arabinoside (arabinofuranosylcytosine)

ASK1 apoptosis signal-regulating kinase-1

ATF-2 activating transcription factor-2

ATP adenosine triphosphate

Bcl-2 B cell lymphoma-2

Bax Bcl Associated X Protein

Bad BAD, Bcl-2/Bcl-XL-antagonist, causing cell death

Bid BH3 interacting death domain agonist

Bim Bcl-2 Interacting Mediator of Cell Death

bp base pairs

BSA bovine serum albumin (albumin fraction V)

cAMP cyclic adenosine monophospate

caspase cysteine aspartate-specific protease

cDNA complementary DNA

c-Fos v-fos FBJ murine osteosarcoma viral oncogene homologue

c-Jun v-Jun avian sarcoma virus 17 oncogene homologue

[Ca2+]i intracellular calcium concentration

CNS central nervous system

COX cyclo-oxygenase

Cu/Zn-SOD copper zinc-SOD

Cyt c cytochrome c

DA dopamine

DAT dopamine transporter

DAB diamino-benzidine

DCF 2’,7’-dichlorofluorescein

DDC DOPA decarboxylase

DDW double-distilled water

DHR dihydrorhodamine

ABBREVIATIONS

159

DIABLO direct IAP binding protein with low pI

DLB denaturing lysis buffer

DMSO dimethylsulfoxide

dn dominant negative

DNA deoxyribonucleic acid

DNAse deoxyribonuclease

dNTP 2’-deoxynucleoside-5’-triphosphate

DOPA 3,4-Dihydroxyphenylalanin

DTT dithiothreitol

ECL enhanced chemiluminescence

EDTA ethylenediaminetetraacetic acid

EGTA Ethylene-bis(oxyethylenenitrilo)tetraacetic acid

EGF epidermal growth factor

Elk-1 Ets-like gene-1

em. emission wavelength

ERK extracellular signal-regulated kinase

ex. excitation wavelength

FCS fetal calf serum

FI fluorescence intensity

Fig. figure

FITC Fluorescein-isothiocyanat

x g relative centrifugal force (RCF)

H2DCF 2’,7’-dichloro-dihydrofluorescein

HEPES N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid

HRP horseradish peroxidase

HS Horse serum

IC50 inhibitory concentration 50% (concentration leading to 50% inhibition)

IgG immunoglobulin G

IL interleukin

IMM inner mitochondrial membrane

IMS mitochondrial intermembrane space

IR immunoreactivity

IU international unit(s)

JIP c-Jun N-terminal kinase-interacting protein

JNK c-Jun N-terminal kinase

ABBREVIATIONS

160

kb kilobase

kDa Kilodalton

KM Michaelis-Menten constant

k.o. knock-out

LB Luria-Bertani medium

LDH Lactate dehydrogenase

MAP mitogen-activated protein

MAPK mitogen-activated protein kinase

MAPKK MAP2K, MEK, MKK, mitogen-activated protein kinase kinase

MAP3K MAPKKK, MEKK, mitogen-activated protein kinase kinase kinase

MCA middle cerebral artery

MEK mitogen-activated protein / extracellular signal-regulated kinase kinase

MEKK mitogen-activated protein / extracellular signal-regulated kinase kinase kinase

MFB medial forebrain bundle

MKK mitogen-activated protein kinase kinase

MLK mixed-lineage kinase

Mn-SOD manganese SOD

MPP+ 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion

MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

mPTP mitochondrial permeability transition pore

mRNA messenger RNA

MTred mitotracker red

N number of independent experiments per experimental series

NFAT nuclear factor of activated T cells

NGF nerve growth factor

N-terminal amino-terminal

P probability

p. a. pro analysis

PAGE polyacrylamide gel electrophoresis

PARP poly(ADP-ribose) polymerase

PBS phosphate-buffered saline

PBST PBS containing Triton X-100

PCR polymerase chain reaction

PD Parkinson’s disease

pH potentia hydrogenii (hydrogen ion concentration)

ABBREVIATIONS

161

PKC protein kinase C

PMSF phenyl-methyl sulfonyl fluoride

PVDF polyvinylidene difluoride

RNA ribonucleic acid

RNAse ribonuclease

ROI region of interest

ROS reactive oxygen species

rpm rotations per minute (centrifuge parameter)

RPMI-1640 Roswell Park Memorial Institute culture medium 1640

RT room temperature (ca. 20°C)

SAPK stress-activated protein kinase

SDS sodium dodecyl sulfate

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEK JNK kinase

Smac second mitochondria-derived activator of caspase

SNpc Substantia Nigra pars compacta

SOD superoxide dismutase

SP600125 anthra(1,9-cd)pyrazol-6(2H)-one

STS staurosporine

Taq Thermus aquaticus

TBE Tris-boric acid-EDTA buffer

TBS Tris-buffered saline

TBST TBS with Tween-20

TEMED N,N,N’,N’-tetramethylethylenediamine

TM melting / annealing temperature of primers

TNF-alpha tumour necrosis factor-alpha

Tris tris-(hydroxymethyl)-aminomethane

Trx thioredoxin

w/o without

w/v weight per volume

DANKSAGUNG/ACKNOWLEDGEMENTS

162

8. DANKSAGUNG/ACKNOWLEDGEMENTS

Ich möchte mich herzlich bedanken bei

I wish to express my gratitude towards

Meinem Doktorvater Prof. Dr. Thomas Herdegen für seine große Unterstützung bei der Durchführung dieser Arbeit, für sein Vertrauen in mich und für seine motivierende und inspirierende Art innerhalb und außerhalb der Forschung.

Meinem Betreuer Prof. Dr. Eric Beitz für die Korrektur meiner Dissertation, für wertvolle Ratschläge und die freundliche und unkomplizierte Übernahme meiner Betreuung.

PD Dr. Mario Götz für die freundschaftliche und fruchtbare Zusammenarbeit, und seine fachliche Beratung.

Desweiteren aus der Pharmakologie in Kiel all meinen Kollegen und Kolleginnen, im Besonderen der besten Bürokollegin Sevgi Eminel, Zhao Yi, Elke Schröder und Inga Wohlers für die immer sehr gute Zusammenarbeit; Vicki und Annika für ihre wertvolle Hilfe und ihren Rat, sowie Tom, Matze und Katja aus der Nephrologie für ideelle Unterstützung.

Prof. Dr. Jari Koistinaho and his group in Kuopio, Finland, for the possibility to learn and to conduct my experiments in his lab.

Meinen Freunden, die mir immer zur Seite gestanden haben, im Besonderen Robert und Lucy, Yun-Bon und Christian.

My friends from around the world, in particular Otto and Terhi, my cat mother Veera, Joana, Kiran, Juliane and Eva.

Conni, Fiffi, Meike und Malin für Eure Unterstützung.

Christine, for your endurance and your faith.

LEBENSLAUF/CURRICULUM VITAE

163

9. LEBENSLAUF/CURRICULUM VITAE

Persönliche Daten

Name: Lutz Römer

Geburtsdatum: 21.12.1976

Geburtsort: Marl

Staatsangehörigkeit: deutsch

Schulausbildung 1987 – 1989 Städtisches Gymnasium Haltern, Haltern

1989 – 1996 Clemens-Brentano-Gymnasium, Dülmen

1996 Allgemeine Hochschulreife

Ersatzdienst 1996 – 1997 Betreuung behinderter Kinder, Regenbogen e.V., Dülmen

Studium und Praktisches Jahr WS 1997 – SS 2002 Studium der Pharmazie an der Christian-Albrechts-Universität

zu Kiel

Juni 2002 – Dez. 2002 Pharmaziepraktikum in der Anker-Apotheke in Kiel

Jan. 2003 – Aug. 2003 Pharmaziepraktikum im Institut für Pharmakologie, UK-SH

(Universitätsklinikum Schleswig-Holstein), Campus Kiel

20. August 2003 Erteilung der Approbation

Promotion und Weiterbildung Sept. 2003 – Feb. 2008 Wissenschaftlicher Mitarbeiter am Institut für Pharmakologie,

UK-SH, Campus Kiel. Anfertigung einer Dissertation unter

Leitung von Prof. Dr. Thomas Herdegen. Forschungsaufenthalt

in Kuopio, Finnland (Prof. Dr. Jari Koistinaho, März 2006 bis

Juni 2007).

seit Juli 2003 Weiterbildung zum Fachapotheker für Arzneimittelinformation,

Betreuung Prof. Dr. Walter Raasch, Institut für Pharmakologie,

UK-SH, Campus Lübeck

LEBENSLAUF/CURRICULUM VITAE

164

Publikationen Eminel S, Roemer L, Waetzig V, Herdegen T (2008). c-Jun N-terminal kinases trigger both

degeneration and neurite outgrowth in primary hippocampal and cortical neurons. Journal of

Neurochemistry. Feb;104(4):957-69.

Wruck CJ, Claussen M, Fuhrmann G, Römer L, Schulz A, Pufe T, Waetzig V, Peipp M,

Herdegen T, Goetz ME (2007). Flavonoid mediated neuroprotection requires activation of the

transcription factor nuclear factor E2-related factor 2 (Nrf-2). Journal of Neural Transmission

Supplement.(72):57-67.

Wessig J, Brecht S, Claussen M, Roemer L, Goetz M, Bigini P, Schutze S, Herdegen T.

(2005). Tumor necrosis factor-alpha receptor 1 (p55) knockout only transiently decreases the

activation of c-Jun and does not affect the survival of axotomized dopaminergic nigral

neurons. European Journal of Neuroscience. Jul;22(1):267-72.

Erninel S, Klettner A, Roemer L, Herdegen T, Waetzig V (2004). JNK2 translocates to the

mitochondria and mediates cytochrome c release in PC12 cells in response to 6-hydroxy-

dopamine. Journal of Biological Chemistry. Dec 31;279(53):55385-92.

Tagungsbeiträge Roemer L, Goldsteins G, Koistinaho J. Generation of reactive oxygen species and

mitochondrial impairment following 6-hydroxydopamine. Oral presentation and abstract.

Graduate School of Molecular Medicine – Winter School 2007. Vuokatti, Finland, 2007.

Eminel S, Roemer L, Klettner A, Herdegen T. JNK2 translocates to mitochondria and

mediates cytochrome c release following 6-hydroxydopamine. Abstract and poster

presentation. 30th Göttingen Neurobiology Conference. 2005.

ERKLÄRUNG ZU §10 ABS. 2 NR. 2 DER PROMOTIONSORDNUNG

165

10. ERKLÄRUNG ZU §10 ABS. 2 NR. 2 DER PROMOTIONSORDNUNG

Der Inhalt dieser Abhandlung wurde, abgesehen von der Beratung durch meinen Betreuer,

selbstständig von mir erarbeitet und in dieser Form zusammengestellt. Die Arbeit hat an

keiner anderen Stelle im Rahmen eines Prüfungsverfahrens vorgelegen.

Kiel, im März 2008

Lutz Römer

SUMMARY

166

11. SUMMARY

Parkinson’s disease (PD) is a progressive neurodegenerative disorder of less defined etiology

and limited treatment options. An important role in the development of the disease has been

addressed to oxidative stress, mitochondrial impairment and stress kinase signaling. The c-Jun

N-terminal kinases (JNK) belong to a subgroup of the mitogen-activated protein

kinases/stresskinases. Togther with their upstream kinases and downstream effectors JNK

compose the JNK signaling pathway. We used PC12 cells from rat pheochromocytoma, a

model for catecholaminergic cells, together with the addition of 6-hydroxydopamine

(6-OHDA), which is a well-established model toxin to induce PD-like symptoms in vivo and

in vitro.

The results of this thesis demonstrate that production of reactive oxygen species (ROS) is

increased shortly after administration of 6-OHDA (60 min). These ROS appear to derive from

mitochondria, which are the greatest intracellular source of ROS if a leakage of protons

occurs; we could also detect an immediate effect of 6-OHDA (or its metabolism products) on

the mitochondrial membrane potential.

Furthermore, we discovered that a single JNK isoform (JNK2) translocates to mitochondria

after stimulus. The signalosome was present at the mitochondria within 4 h, and it consisted

of presumably activated JNK2, the upstream activator MKK4 and the scaffold protein for

both, JIP. 6-OHDA also lead to a release of cytochrome c from the mitochondrial

intermembrane space into the cytosol starting 8 h following 6-OHDA. This indicates

disturbances of the outer mitochondrial membrane, formation of pores, and the cytosolic

activation of caspases, which finally lead to cell death after 24 h.

JNK inhibition prevents various features of JNK pathology such as activation, translocation to

the mitochondria, cytochrome c release and cell death, but not from ROS generation. PC12

cells could only be protected from this early event, upstream of JNK, by preincubation of

plant-derived antioxidants, that also increase cellular defense mechanisms.

Our data confirm that JNK exhibit dual involvement in cell death and survival, as inhibition

of JNK attenuates neuritogenesis of primary hippocampal neurons severely, regardless of the

isoform. Therefore, the perspective of therapeutic JNK inhibition has to be reconsidered.

ZUSAMMENFASSUNG

167

12. ZUSAMMENFASSUNG

Der Morbus Parkinson ist eine progressiv-degenerative Erkrankung des Gehirns mit derzeit

limitierten Behandlungsoptionen, dessen Aetiologie noch nicht eindeutig beschrieben werden

konnte. Eine wichtige Rolle in der Krankheitsentstehung spielen oxidativer Stress,

Beeinträchtigung der Mitochondrienfunktion und intrazelluläre Signalkaskaden vermittelt von

Stresskinasen. Die c-Jun N-terminalen Kinasen (JNK) gehören zu einer Untergruppe dieser

Mitogen-aktivierten Proteinkinasen/Stresskinasen. JNK bilden zusammen mit ihren

Upstream-Kinasen und Downstream-Zielstrukturen den JNK Signalweg. Wir wählten die

permanente Zelllinie PC12 aus dem Rattenphäochromocytom, ein Modell einer zentralen

catecholaminergen Zelllinie, und behandelten diese mit 6-Hydroxydopamin (6-OHDA), ein

Toxin, welches zur Darstellung von Parkinson-ähnlichen Symptomen in vivo und in vitro

eingesetzt wird.

Die Ergebnisse dieser Arbeit zeigen, daß 6-OHDA die Produktion reaktiver Sauerstoffspezies

(ROS) innerhalb kurzer Zeit stark erhöht (60 min). Die ROS wurden in Mitochondrien

gebildet, der größten Quelle für Sauerstoffradikale, sofern das Membranpotential nicht stabil

ist und Protonen in der intermembranären Zwischenraum gelangen können. Wir beobachteten

ferner einen sofortigen Effekt von 6-OHDA (oder dessen Metabolisationsprodukten) auf das

mitochondriale Membranpotential.

Desweiteren fanden wir heraus, daß eine einzige JNK Isoform (JNK2) nach Gabe des

Stimulus 6-OHDA zu den Mitochondrien transloziert. Das gesamte Signalosom aus

aktiviertem JNK2, der Upstream-Kinase MKK4, welche verantwortlich für die Aktivierung

von JNK ist, und dem Scaffold-Protein JIP findet sich an den Mitochondrien innerhalb von

4 Stunden nach Stiumulus. Außerdem führte die Zugabe von 6-OHDA zu einer Freisetzung

von Cytochrom C aus den Mitochondrien ins Zytosol, beginnend nach etwa 8 Stunden. Dies

deutet klar auf eine eingeschränkte Funktion der äußeren Mitochondrienmembran, der

Bildung von Poren und der Aktivierung von Caspasen im Zytosol hin. Nach 24 Stunden

beobachteten wir dann einen deutlichen Zelltod in vitro im Anschluß an die Freisetzung von

Cytochrom C.

JNK Inhibierung bewahrt die Zelle vor verschiedenen apoptotischen Effekten der

beschriebenen Pathologie, wie der JNK-Aktivierung, der Translokation zu den

ZUSAMMENFASSUNG

168

Mitochondrien, Cytochrom-C-Freisetzung und vor Zelltod, jedoch nicht vor oxidativem

Stress. PC12 Zellen konnten davor nur geschützt werden, wenn vor der Toxingabe mit einem

pflanzlichen Antioxidans (Methysticin, Luteolin, Resveratrol; und tert-Butylhydroquinon)

behandelt wurde, ein Hinweis, daß ROS upstream von JNK sind.

Unsere Ergebnisse bestätigen, daß JNKs eine Rolle sowohl im neuronalen Zelltod, als auch

dem Überleben spielen, da wir mit der Inhibierung von JNK die Neuritogenese von primären

hippokampalen Neuronen stark beeinträchtigen, unabhängig von der Isoform. Aus diesem

Grunde muß die JNK-Inhibition mit SP600125 als potentieller Therapieansatz beim Morbus

Parkinson überdacht werden.

Documents

Pro- and antidegenerative effects of JNK stresskinases in ... · 4.3.2 Activation of the mitochondrial pool of JNK following 6-OHDA 98 4.3.3 Translocation of c-Jun N-terminal kinase